http://2011.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=250&target=Hargem&year=&month=2011.igem.org - User contributions [en]2024-03-29T11:44:06ZFrom 2011.igem.orgMediaWiki 1.16.0http://2011.igem.org/Team:Washington/Alkanes/Future/VectorTeam:Washington/Alkanes/Future/Vector2011-11-20T20:27:27Z<p>Hargem: </p>
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<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
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='''<center>System Optimization</center>'''=<br />
<br />
Using our initial, non-optimized growth conditions, we were able to obtain alkane/ene yields of approximately 2 mg/L. In order to make analysis easier, and to make it easier to determine the effects of the addition of additional modules, we wanted to increase yield. Our efforts focused on how by varying system conditions, we could increase yield.<br />
<br />
=='''Sealed vs. Open Tubes'''==<br />
We were concerned that produced alkanes may have been evaporating, reducing apparent yield. Therefore, we performed tests where the tubes were either capped the standard way, or capped with foil coverting the opening, thereby reducing evaporation. Tests were conducted in glass tubes with M9 production media, with MG1655 innoculated to an initial OD600 of 1.<br />
[[File:Washinton_open_sealed.png|center|500px|thumb|Sealing Culture Tubes Increases Yield.]]<br />
Covered tubes showed significantly more alkanes, so all further tests would be conducted in sealed tubes.<br />
<br />
=='''Use of Different Strains'''==<br />
We had suspected that different strains of ''E. coli'' would produce varying amounts of alkanes. Initial experiments were done in MG1655, and we decided to test XL-1 blue (a commercial supercopentent variant of DH5a) for the ability to produce alkanes. Tests were conducted in sealed glass culture tubes in M9 production media . Cells were innoculated to an OD600 of 1 when conducting this test.<br />
[[File:Washinton_XL21vsMG1655.png|center|500px|thumb|XL-1 Blue Produces more Alkane than MG1655]]<br />
XL-1 blue was able to produce more alkanes than MG1655. Therefore, future tests were conducted in XL-1 Blue.<br />
<br />
=='''Aerobic vs Microaerobic Growth'''==<br />
In many industrial production applications, growth in conditions with little or no oxygen can improve yield. Therefore, we tested alkane yield in cultures grown in airtight vials with only a small amount of air on the top in order to severely limit oxygen availability. Tests were conducted in glass (note that the test was conducted in open culture tubes for aerobic cultures, and sealed glass vials for microaerobic cultures, with M9 production media in XL21-blue cells.)<br />
[[File:Washington_aerobicVsMicroaerobic.png|center|500px|thumb|Oxygen has a Positive Effect on Alkane Production]]<br />
Based upon this data, aerobic growth appears to be better for alkane yield. Note that this difference is not likely due to differences in growth rate, as both cultures were inoculated to a high OD600 of 1. The fact that this tube was not covered means that the actual alkane yield (before evaporation) was likely higher. Therefore, microaerobic conditions are unlikely to improve yield.<br />
<br />
=='''pSB1C3-ADC-AAR vs pSB3k3-ADC/pSB4C5-AAR'''==<br />
The original study expressed ADC and AAR on seperate low copy number vectors. If we could get high alkane yields using low copy number vectors, the lesser protien and vector production would make the use of a lower copy number vector preferable. Tests were conducted in covered glass culture tubes (aerobic conditions) with M9 production media. Cells were XL1-Blue, and were innoculated to an OD600 of 1.<br />
[[File:Washington_copynumber.png|center|500px|thumb|Greater ADC/AAR Expression Results in More Alkane Production.]]<br />
The fact that high copynumber vector use results in an increase in yield means that high expression levels of ADC and/or AAR are required for high alkane yields.<br />
<br />
=='''Media composition'''==<br />
Our initial media was based upon the media used in [[#References |[1]]]. We did not know if all of the additional components not found in a normal media (buffer, thiamine, Triton, iron) actually improved yield. In order to determine if each of these components imporved yield, we analyzed cultures inoculated into M9 medias without each of these 4 compounds. In addition, we tested media with additional trace metals. These tests were conducted in covered glass culture tubes with XL-1 blue cells.<br />
[[File:Washington_mediaconditions.png|center|500px|thumb|Effects of Media Composition on Alkane Yield.]]<br />
None of the modifications we made resulted in a significant increase in yield. Therefore, no further modifications to the media used was made.<br />
<br />
=='''Effects of Changing Initial Cell Density'''==<br />
Earlier tests were done with an initial OD600 of 1. However, we had never established what the optimal initial OD would be. If significant alkane production occurs during stationary phase, inoculation to a high initial OD may increase alkane production. For this test, we inoculated XL-1 blue (to a final OD600 in a range of 0.01 to 10) in M9 production media (in covered glass culture tubes). We extracted from these cultures after 16 hours of growth. <br />
[[File:Washington_alkaneODcurve.png|center|500px|thumb|Greater Initial Cell Density Results in Higher Alkane Yield.]]<br />
Higher cell density cultures showed significantly higher alkane yields than lower cell density cultures. The increase of alkane yields at cell densities as high as 10 implies that high amounts of alkane production occurs during stationary phase. Therefore, alkane production scales with cell density. In addition, inducing alkane production during stationary phase decreases the amount of glucose going towards cellular growth instead of alkane production.<br />
=='''Final conditions'''==<br />
Using our optimized media & growth conditions(XL-1 blue inoculated to an OD600 of 10, in M9 media, in closed glass culture tubes) , we were able to improve total alkane yield 80 fold, from approximenly 2 mg/L (after 48 hrs) to approximently 170 mg/L(after 48 hrs).<br />
[[File:Washinton 2011 Optimization Quant.png|center|550px|thumb|Our optimized growth conditions resulted in an 80 fold increase in total alkane yield.]]<br />
=='''Production Curve'''==<br />
Ideally, alkane extraction and analysis would occur after all ( or essentially all) of the glucose in the media was used up. However, we didn't know how long it would take for our cells to use up all of the glucose. In order to determine the best time for extraction, we extracted alkanes from M9 production cultures that had been producing for 6, 24, 48, and 72 hours. Three cultures were used for each timepoint.<br />
[[file:Washington_Productioncurve.png|center|550px|thumb|Total alkane yield over time under optimized conditions.]]<br />
The 24 hour timepoint was essentially identical to the 48 and 72 hour timepoints. This implies that our cells are using up 30g/L glucose sometime before 24 hours. Future work will consist of increasing glucose concentration, and increasing inoculated cell density in order to maximize alkane production per culture per day.<br />
=='''Alternate Carbon Sources'''==<br />
While previous tests were done using glucose as the sole carbon source, it would be helpful to be able to use carbon sources that can be considered waste sources. There is a large amount of interest in using non-simple sugar carbon sources, such as cellulosic biomass, and even using carbon dioxide directly. However, these carbon sources cannot be easily utilized by ''E. coli''. Glycerol is a 3-carbon triol that is a waste product in the production of current generation biodiesel(fatty acid methyl esters(FAMEs). Glycerol can be used by ''E. coli'' as a sole carbon source, so we tried to use the PetroBrick to convert glycerol into diesel. We performed standard alkane production/analysis, with the second production media being either [https://2011.igem.org/Team:Washington/alkanebiosynthesis#Current_Protocol_for_100mL_of_Media standard M9 glucose media], or M9 media varients containing an equal mass of glycerol instead of glucose, or media without any carbon source.<br />
[[File:Washington_carbontest.png|center|550px|thumb|Total alkane yield in M9 glucose, M9 glycerol, and M9 nedia without carbon.]]<br />
The glucose media resulted in higher total alkane yield than the glycerol media(240 mg/L vs. 56.3 mg/L), but this may be due to media conditions being optimized for glucose as a carbon source insteam od glycerol. Further optimization may be able to improve on alkane yield when using glycerol as a carbon source. In media without carbon, we observed negligable alkane production(4.7 mg/L). This indicates that alkane production is due to conversion of carbon in production media into alkanes, not due to the conversion of cellular biomass into alkanes. This means that initial growth for cell density can occur in a rich media without having any major influence on yield. <br />
=='''References'''==<br />
Microbial Biosynthesis of Alkanes Andreas Schirmer, Mathew A. Rude, Xuezhi Li, Emanuela Popova and Stephen B. del Cardayre Science 30 July 2010: Vol. 329 no. 5991 pp. 559-562 http://www.sciencemag.org/content/329/5991/559</div>Hargemhttp://2011.igem.org/World_Championship_Jamboree/Schedule/Practice_SessionsWorld Championship Jamboree/Schedule/Practice Sessions2011-11-02T14:23:07Z<p>Hargem: </p>
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<th>RM 56-162 </th><br />
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<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Future Directions'''</big></big></big></big></center><br><br><br />
<br />
Our current ''in vivo'' alkane production system efficiently makes C15 alkanes. However, to be efficient enough for factory production, there are two broad goals to be done: <br />
# Increase production efficiency <br />
# Increase the diversity of the range of alkanes that can be produced. <br />
# Increase the scale of the system for industrial processes. <br />
We have already begun efforts to expand the efficiency and scope of alkane production, '''check them out below!'''<br />
<br />
[[Image:UW_2011_Alkane_Future_Work_Image.png|7px||right]]<br />
<br />
[[Image:Washington system optimization.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Vector]]<br />
<br><br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Vector '''System Optimization''']<br />
:<nowiki> The efficiency of this system was reported to be much higher than our initial system, but the growth conditions of the assay and the DNA was not available to us. We increased production efficiency by altering the initial environmental conditions in the assay.</nowiki><br />
<br><br><br />
<br />
[[Image:Washington_2011_ADC_Redesign.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/DecarbDesign]]<br />
<br><br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/DecarbDesign '''Decarbonylase Redesign''']<br />
:<nowiki>One way to diversify the kind of alkanes produced is to alter the substrate specificity of the proteins involved. Since an alternative alkane-producing enzyme has not been found, we decided to mutate the aldehyde decarbonylase to produce shorter-chain alkanes.</nowiki><br />
<br><br><br />
<br />
[[Image:Washington_2011_LuxCDE.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/LuxCDE]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/LuxCDE '''Alternative Aldehyde''']<br />
:<nowiki>Another way to diversify our system is to use alternative proteins. Our current system uses acyl-ACP reductase, and we've identified an hypothetical alternative system that produces aldehydes: LuxCDE, made from parts from the 2010 competition.</nowiki><br />
<br><br><br><br />
<br />
[[Image:Washington_2011_fabh2_branch.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2 '''Alternitive Alkane Products''']<br />
:<nowiki> The PetroBrick is only capable of producing unbranched, odd chain length alkanes, as the cell mainly utilizes straight chained, even chain length fatty acids. However, fuel we use is composed of a wide range of products. By changing which fatty acids are made by alkane producing cells, we could theoretically change which alkanes are being produced by the PetroBrick. </nowiki><br />
<br><br />
<br />
[[Image:Washington 2011 Protein Localization.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Localization]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Localization '''Enzyme Localization''']<br />
:<nowiki> The easiest way to increase yield is to increase the concentration; if the number of molecules remained the same but the volume decreased, the reaction speed increases with occurrences of molecular collisions. However, this is only easily done with purified protein assays. However, there are ways to "increase the concentration" of reactions in cells. We cannot make the cells literally denser, but we can localize the enzymes involved in the reaction, which either decreases the volume of the cell in which the enzymes reside and/or limit the number of reaction steps.</nowiki><br />
<br />
[[Image:Washington_2011_Alternate_Chassis.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Yeast]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Yeast '''Alternative Chassis''']<br />
:<nowiki> By synthesizing alkanes in organisms, alkanes can become a useful renewable energy source; CO2 emitted from burning the alkanes are converted to glucose through photosynthesis, and then the glucose can be fed to alkane-synthesizing organisms to restart the cycle. This cycle can be drastically improved if there were fewer steps in the cycle. We explored the possibility of optimizing the alkane biosynthesis system in different micro-organisms, with the idea that perhaps with autotrophic organisms, we do not need to obtain glucose for the reaction.</nowiki><br />
<br />
<br />
<br />
[[Image:Washington_FB.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Modeling]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Modeling '''System Modeling''']<br />
:<nowiki> We have begun Flux Balance Modeling the alkane production system in order to better understand the carbon flux through the pathway. We believe that by understanding the curent flux through the pathway we can intelligently reengineer the platform in order to maximize flux to alkanes. </nowiki></div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/FutureTeam:Washington/Alkanes/Future2011-10-29T00:34:21Z<p>Hargem: Undo revision 258777 by Hargem (talk)</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Future Directions'''</big></big></big></big></center><br><br><br />
<br />
Our current ''in vivo'' alkane production system efficiently makes C15 alkanes. However, to be efficient enough for factory production, there are two broad goals to be done: <br />
# Increase production efficiency <br />
# Increase the diversity of the range of alkanes that can be produced. <br />
# Increase the scale of the system for industrial processes. <br />
We have already begun efforts to expand the efficiency and scope of alkane production, '''check them out below!'''<br />
<br />
[[Image:UW_2011_Alkane_Future_Work_Image.png|7px||right]]<br />
<br />
[[Image:Washington system optimization.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Vector]]<br />
<br><br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Vector '''System Optimization''']<br />
:<nowiki> The efficiency of this system was reported to be much higher than our initial system, but the growth conditions of the assay and the DNA was not available to us. We increased production efficiency by altering the initial environmental conditions in the assay.</nowiki><br />
<br><br><br />
<br />
[[Image:Washington_2011_ADC_Redesign.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/DecarbDesign]]<br />
<br><br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/DecarbDesign '''Decarbonylase Redesign''']<br />
:<nowiki>One way to diversify the kind of alkanes produced is to alter the substrate specificity of the proteins involved. Since an alternative alkane-producing enzyme has not been found, we decided to mutate the aldehyde decarbonylase to produce shorter-chain alkanes.</nowiki><br />
<br><br><br />
<br />
[[Image:Washington_2011_LuxCDE.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/LuxCDE]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/LuxCDE '''Alternative Aldehyde''']<br />
:<nowiki>Another way to diversify our system is to use alternative proteins. Our current system uses acyl-ACP reductase, and we've identified an hypothetical alternative system that produces aldehydes: LuxCDE, made from parts from the 2010 competition.</nowiki><br />
<br><br><br><br />
<br />
[[Image:Washington_2011_fabh2_branch.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2 '''Branched Alkane Biosynthesis''']<br />
:<nowiki> Our system is only capable of producing unbranched alkanes, as the cell mainly utilizes straight chained fatty acids. However, fuel we use are also composed largely of branched alkanes that affect very important properties of the fuel such as flash point and freezing point. If our fuels are truly intended to be synthesized in bacteria, we need to work on methods of making those crucial branched chained alkanes. We explored FabH2, a protein that when involved in fatty acid synthesis makes branched fatty acids. </nowiki><br />
<br><br />
<br />
[[Image:Washington 2011 Protein Localization.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Localization]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Localization '''Enzyme Localization''']<br />
:<nowiki> The easiest way to increase yield is to increase the concentration; if the number of molecules remained the same but the volume decreased, the reaction speed increases with occurrences of molecular collisions. However, this is only easily done with purified protein assays. However, there are ways to "increase the concentration" of reactions in cells. We cannot make the cells literally denser, but we can localize the enzymes involved in the reaction, which either decreases the volume of the cell in which the enzymes reside and/or limit the number of reaction steps.</nowiki><br />
<br />
[[Image:Washington_2011_Alternate_Chassis.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Yeast]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Yeast '''Alternative Chassis''']<br />
:<nowiki> By synthesizing alkanes in organisms, alkanes can become a useful renewable energy source; CO2 emitted from burning the alkanes are converted to glucose through photosynthesis, and then the glucose can be fed to alkane-synthesizing organisms to restart the cycle. This cycle can be drastically improved if there were fewer steps in the cycle. We explored the possibility of optimizing the alkane biosynthesis system in different micro-organisms, with the idea that perhaps with autotrophic organisms, we do not need to obtain glucose for the reaction.</nowiki><br />
<br />
<br />
<br />
[[Image:Washington_FB.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Modeling]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Modeling '''System Modeling''']<br />
:<nowiki> We have begun Flux Balance Modeling the alkane production system in order to better understand the carbon flux through the pathway. We believe that by understanding the curent flux through the pathway we can intelligently reengineer the platform in order to maximize flux to alkanes. </nowiki></div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/FutureTeam:Washington/Alkanes/Future2011-10-29T00:34:01Z<p>Hargem: Undo revision 258825 by Hargem (talk)</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Future Directions'''</big></big></big></big></center><br><br><br />
<br />
Our current ''in vivo'' alkane production system efficiently makes C15 alkanes. However, to be efficient enough for factory production, there are two broad goals to be done: <br />
# Increase production efficiency <br />
# Increase the diversity of the range of alkanes that can be produced. <br />
# Increase the scale of the system for industrial processes. <br />
We have already begun efforts to expand the efficiency and scope of alkane production, '''check them out below!'''<br />
<br />
[[Image:UW_2011_Alkane_Future_Work_Image.png|7px||right]]<br />
<br />
[[Image:Washington system optimization.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Vector]]<br />
<br><br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Vector '''System Optimization''']<br />
:<nowiki> The efficiency of this system was reported to be much higher than our initial system, but the growth conditions of the assay and the DNA was not available to us. We increased production efficiency by altering the initial environmental conditions in the assay.</nowiki><br />
<br><br><br />
<br />
[[Image:Washington_2011_ADC_Redesign.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/DecarbDesign]]<br />
<br><br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/DecarbDesign '''Decarbonylase Redesign''']<br />
:<nowiki>One way to diversify the kind of alkanes produced is to alter the substrate specificity of the proteins involved. Since an alternative alkane-producing enzyme has not been found, we decided to mutate the aldehyde decarbonylase to produce shorter-chain alkanes.</nowiki><br />
<br><br><br />
<br />
[[Image:Washington_2011_LuxCDE.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/LuxCDE]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/LuxCDE '''Alternative Aldehyde''']<br />
:<nowiki>Another way to diversify our system is to use alternative proteins. Our current system uses acyl-ACP reductase, and we've identified an hypothetical alternative system that produces aldehydes: LuxCDE, made from parts from the 2010 competition.</nowiki><br />
<br><br><br><br />
<br />
[[Image:Washington_2011_fabh2_branch.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2 '''Alternative Alkane Products ''']<br />
:<nowiki> Our system is only capable of producing unbranched, odd chain length alkanes, as the cell mainly utilizes straight chained, even chain length fatty acids. However, fuel we use is composed of a wide range of products. By changing which fatty acids are made by alkane producing cells, we could theoretically change which alkanes are being produced by our system.<br />
<br><br />
<br />
[[Image:Washington 2011 Protein Localization.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Localization]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Localization '''Enzyme Localization''']<br />
:<nowiki> The easiest way to increase yield is to increase the concentration; if the number of molecules remained the same but the volume decreased, the reaction speed increases with occurrences of molecular collisions. However, this is only easily done with purified protein assays. However, there are ways to "increase the concentration" of reactions in cells. We cannot make the cells literally denser, but we can localize the enzymes involved in the reaction, which either decreases the volume of the cell in which the enzymes reside and/or limit the number of reaction steps.</nowiki><br />
<br />
[[Image:Washington_2011_Alternate_Chassis.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Yeast]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Yeast '''Alternative Chassis''']<br />
:<nowiki> By synthesizing alkanes in organisms, alkanes can become a useful renewable energy source; CO2 emitted from burning the alkanes are converted to glucose through photosynthesis, and then the glucose can be fed to alkane-synthesizing organisms to restart the cycle. This cycle can be drastically improved if there were fewer steps in the cycle. We explored the possibility of optimizing the alkane biosynthesis system in different micro-organisms, with the idea that perhaps with autotrophic organisms, we do not need to obtain glucose for the reaction.</nowiki><br />
<br />
<br />
<br />
[[Image:Washington_FB.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Modeling]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Modeling '''System Modeling''']<br />
:<nowiki> We have begun Flux Balance Modeling the alkane production system in order to better understand the carbon flux through the pathway. We believe that by understanding the curent flux through the pathway we can intelligently reengineer the platform in order to maximize flux to alkanes. </nowiki></div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/FutureTeam:Washington/Alkanes/Future2011-10-29T00:33:39Z<p>Hargem: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Future Directions'''</big></big></big></big></center><br><br><br />
<br />
Our current ''in vivo'' alkane production system efficiently makes C15 alkanes. However, to be efficient enough for factory production, there are two broad goals to be done: <br />
# Increase production efficiency <br />
# Increase the diversity of the range of alkanes that can be produced. <br />
# Increase the scale of the system for industrial processes. <br />
We have already begun efforts to expand the efficiency and scope of alkane production, '''check them out below!'''<br />
<br />
[[Image:UW_2011_Alkane_Future_Work_Image.png|7px||right]]<br />
<br />
[[Image:Washington system optimization.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Vector]]<br />
<br><br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Vector '''System Optimization''']<br />
:<nowiki> The efficiency of this system was reported to be much higher than our initial system, but the growth conditions of the assay and the DNA was not available to us. We increased production efficiency by altering the initial environmental conditions in the assay.</nowiki><br />
<br><br><br />
<br />
[[Image:Washington_2011_ADC_Redesign.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/DecarbDesign]]<br />
<br><br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/DecarbDesign '''Decarbonylase Redesign''']<br />
:<nowiki>One way to diversify the kind of alkanes produced is to alter the substrate specificity of the proteins involved. Since an alternative alkane-producing enzyme has not been found, we decided to mutate the aldehyde decarbonylase to produce shorter-chain alkanes.</nowiki><br />
<br><br><br />
<br />
[[Image:Washington_2011_LuxCDE.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/LuxCDE]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/LuxCDE '''Alternative Aldehyde''']<br />
:<nowiki>Another way to diversify our system is to use alternative proteins. Our current system uses acyl-ACP reductase, and we've identified an hypothetical alternative system that produces aldehydes: LuxCDE, made from parts from the 2010 competition.</nowiki><br />
<br><br><br><br />
<br />
[[Image:Washington_2011_fabh2_branch.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2 '''Alternative Alkane Products ''']<br />
:<nowiki> Our system is only capable of producing unbranched, odd chain length alkanes, as the cell mainly utilizes straight chained, even chain length fatty acids. However, fuel we use is composed of a wide range of products. By changing which fatty acids are made by alkane producing cells, we could theoretically change which alkanes are being produced by our system.<br />
<br><br />
<br />
[[Image:Washington 2011 ProteinLocalization.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Localization]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Localization '''Enzyme Localization''']<br />
:<nowiki> The easiest way to increase yield is to increase the concentration; if the number of molecules remained the same but the volume decreased, the reaction speed increases with occurrences of molecular collisions. However, this is only easily done with purified protein assays. However, there are ways to "increase the concentration" of reactions in cells. We cannot make the cells literally denser, but we can localize the enzymes involved in the reaction, which either decreases the volume of the cell in which the enzymes reside and/or limit the number of reaction steps.</nowiki><br />
<br />
[[Image:Washington_2011_Alternate_Chassis.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Yeast]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Yeast '''Alternative Chassis''']<br />
:<nowiki> By synthesizing alkanes in organisms, alkanes can become a useful renewable energy source; CO2 emitted from burning the alkanes are converted to glucose through photosynthesis, and then the glucose can be fed to alkane-synthesizing organisms to restart the cycle. This cycle can be drastically improved if there were fewer steps in the cycle. We explored the possibility of optimizing the alkane biosynthesis system in different micro-organisms, with the idea that perhaps with autotrophic organisms, we do not need to obtain glucose for the reaction.</nowiki><br />
<br />
<br />
<br />
[[Image:Washington_FB.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Modeling]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Modeling '''System Modeling''']<br />
:<nowiki> We have begun Flux Balance Modeling the alkane production system in order to better understand the carbon flux through the pathway. We believe that by understanding the curent flux through the pathway we can intelligently reengineer the platform in order to maximize flux to alkanes. </nowiki></div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/ResultsTeam:Washington/Alkanes/Results2011-10-29T00:32:53Z<p>Hargem: /* The FabBrick, a module for even chain length alkane production */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
<center><big><big><big><big>'''Diesel Production: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
='''GCMS confirms The PetroBrick enables diesel production from sugar using ''E. coli'''''=<br />
<br />
We performed GC-MS analysis on extracts from cell cultures expressing both [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 Acyl-ACP Reductase] (AAR) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 Aldehyde Decarbonylase] (ADC) (this composite part is designated [http://partsregistry.org/Part:BBa_K590025 The PetroBrick]). To act as controls and in order to show that the alkane production system is working as expected, we also analyzed cell cultures expressing either only AAR, or only ADC.<br />
<br />
<br />
[[File:Washington PetroBrick.png|300px|frameless|border|left|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
[[Image:Washinton_AARADCGC-MS.png|center|600px|thumb|GCMS confirms PetroBrick diesel production from sugar using E. coli]]<br />
<br />
<br><br />
<br />
'''No significant hydrocarbons peaks in samples extracted from cells expressing only ADC.''' The blue GCMS chromatagram trace on the bottom show no significant peaks are found on the ADC only control within the time range that we expect alkane signal based upon GC-MS runs on chemical standards (8-10.5 minutes). This is expected, as ''E. coli'' does not normally produce any long chain length aldehyde substrates.<br />
<br />
'''C16 alcohols are observed when AAR is expressed on its own.''' The red GCMS chromatagram trace in the middle has a significant peak at 10.2 minutes corresponding to the C16 alcohol (as confirmed by comparison of the peak's MS spectra to a reference library) is observed when AAR is expressed in MG1655 E. coli. The production of C16 alcohols in cells expressing AAR (but not in cells expressing only ADC) is consistent with AAR reducing even chain length Acyl-ACPs into even chain length fatty aldehydes, which are further reduced by aldehyde dehydrogenases to the alcohol.<br />
<br />
'''The PetroBrick Works! C13, C15, and C17 alkanes are produced when both AAR and ADC are expressed.''' <br />
[[File:Washington2011 Spectra.png|right|550px|thumb|Comparasion of the NIST reference C15 alkane spectrum( in blue) to the C15 peak spectrum taken from a PetroBrick culture (in red). Note the strong m/z peak at 212, corresponding to the parent ion of the C15 alkane. For spectra of the C13 alkane and C17 alkene, refer to our [https://2011.igem.org/Team:Washington/Alkanes/spectra Spectra Page.]]]<br />
The green GCMS chromatagram trace in the middle has a significant new peaks corresponding to the C13 (8.2min) and C15 (9.2min) alkanes, as well as a peak at 10.2 minutes that contains both the C14 alcohol and the C17 alkene. The fact that the C17 alkene peak overlaps with the C14 alcohol peak makes exact quantification of C17 akene yields impossible, but we have sufficient evidence to determine that both molecules are present in the 10.2 minute peak( the earlier section of the peak has an MS spectra consistent with the C16 alcohol, and the later section is consistent with the C17 alkene). The C13 and C15 alkane peaks showed MS spectra highly consistent with C13 and C15 alkane reference spectra(for these spectra, as well as the C17 alkene spectrum, refer to our [https://2011.igem.org/Team:Washington/Alkanes/spectra Spectra Page.]). In addition, both alkane molecules eluted off of the GC at the same time as C13 and C15 alkane standards, further increasing confidence that the 8.2 min and 9.2 min peaks do correspond to the C13 and C15 alkanes. Odd chain length alkane production is what is expected from our system, as ADC removes a carbonyl group from even chain length aldehydes produced by AAR, yielding an odd chain length alkane. Using the PetroBrick, we can turn simple sugars into diesel, a fuel fully compatible with modern infrastructure.<br />
<br />
<br />
----<br />
<br />
='''Initial Quantization of Alkane Production'''=<br />
In order to be able to know how much alkane was being produced by our ''E. coli'', we spiked known amounts of alkane into cell cultures known to not produce alkanes. We then extracted using ethyl acetate, and analyzed extracts using GC-MS. Since peak area corresponds to the amount of each substance present, we used these GC plots to make a standard curve that allows us to convert peak area into an absolute yield. To determine how much alkane was being produced by the Petrobrick, we grew up 3 MG1655 cell cultures transformed with the PetroBrick in M9 production media ([https://2011.igem.org/Team:Washington/alkanebiosynthesis link]), and analyzed using GC-MS.<br />
<br />
[[File:Washington_alkanestandardcurve.png|right|450px|thumb|Standard curves for converting peak area to an absolute amount. Note that these curves is almost perfectly linear. In addition, the curve generated from each alkane is nearly identical, allowing us to use 1, average curve for all 3 different alkanes.]]<br />
[[File:Washinton 2011 Pre-Optimization Quant.png|left|450px|thumb|Diagram showing yields of the C13 and C15 alkanes. Note: The C17 alkene is not included due to inability to quantify.]]<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
='''Optimized Production'''=<br />
By optimizing the method of growth, media, vectors, and cell line we were able to successfully increase alkane yield over 80-fold. Note that this improvement doesn't include C17 alkene production, as we were unable to quantify the C17 alkene peak due to co-elution with the C14 alcohol. The parameters we adjusted for optimization are discussed in detail at [https://2011.igem.org/Team:Washington/Alkanes/Future/Vector our systems optimization] page under future directions, as this is an on going process.<br />
<br />
[[File:Washinton 2011 Optimization Quant.png|center|550px|thumb|Our optimized growth conditions resulted in an 80 fold increase in total alkane yield.]]<br />
='''The FabBrick, a module for even chain length alkane production'''=<br />
In order to more closely match the composition of diesel, we wanted a way to make the C14 and C16 alkanes. We found an enzyme, FabH2 cfrom ''Bacillus subtilis'' that has been hypothesized to start fatty acid biosynthesis with a 3 carbon unit instead of the normal 2 carbon unit. We thought that if we could express FabH2 together with the PetroBrick, we could make even chain length alkanes. WE cloned FabH2 into a alc inducible promoter to form [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590064 the FabBrick], an add-on module to teh PetroBrick system. The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/PetroBrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When the FabBrick is induced, we see a new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that of alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinantly, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels. The FabBrick system shows the modularity and expandability of the PetroBrick system, and is a first proof of concept of the expansion of PetroBricks to produce a wider range of products. For more information on FabH2, refer to our [https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2 future directions page ].<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/ResultsTeam:Washington/Alkanes/Results2011-10-29T00:32:40Z<p>Hargem: /* The FabBrick, a module for even chain length alkane production */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
<center><big><big><big><big>'''Diesel Production: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
='''GCMS confirms The PetroBrick enables diesel production from sugar using ''E. coli'''''=<br />
<br />
We performed GC-MS analysis on extracts from cell cultures expressing both [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 Acyl-ACP Reductase] (AAR) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 Aldehyde Decarbonylase] (ADC) (this composite part is designated [http://partsregistry.org/Part:BBa_K590025 The PetroBrick]). To act as controls and in order to show that the alkane production system is working as expected, we also analyzed cell cultures expressing either only AAR, or only ADC.<br />
<br />
<br />
[[File:Washington PetroBrick.png|300px|frameless|border|left|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
[[Image:Washinton_AARADCGC-MS.png|center|600px|thumb|GCMS confirms PetroBrick diesel production from sugar using E. coli]]<br />
<br />
<br><br />
<br />
'''No significant hydrocarbons peaks in samples extracted from cells expressing only ADC.''' The blue GCMS chromatagram trace on the bottom show no significant peaks are found on the ADC only control within the time range that we expect alkane signal based upon GC-MS runs on chemical standards (8-10.5 minutes). This is expected, as ''E. coli'' does not normally produce any long chain length aldehyde substrates.<br />
<br />
'''C16 alcohols are observed when AAR is expressed on its own.''' The red GCMS chromatagram trace in the middle has a significant peak at 10.2 minutes corresponding to the C16 alcohol (as confirmed by comparison of the peak's MS spectra to a reference library) is observed when AAR is expressed in MG1655 E. coli. The production of C16 alcohols in cells expressing AAR (but not in cells expressing only ADC) is consistent with AAR reducing even chain length Acyl-ACPs into even chain length fatty aldehydes, which are further reduced by aldehyde dehydrogenases to the alcohol.<br />
<br />
'''The PetroBrick Works! C13, C15, and C17 alkanes are produced when both AAR and ADC are expressed.''' <br />
[[File:Washington2011 Spectra.png|right|550px|thumb|Comparasion of the NIST reference C15 alkane spectrum( in blue) to the C15 peak spectrum taken from a PetroBrick culture (in red). Note the strong m/z peak at 212, corresponding to the parent ion of the C15 alkane. For spectra of the C13 alkane and C17 alkene, refer to our [https://2011.igem.org/Team:Washington/Alkanes/spectra Spectra Page.]]]<br />
The green GCMS chromatagram trace in the middle has a significant new peaks corresponding to the C13 (8.2min) and C15 (9.2min) alkanes, as well as a peak at 10.2 minutes that contains both the C14 alcohol and the C17 alkene. The fact that the C17 alkene peak overlaps with the C14 alcohol peak makes exact quantification of C17 akene yields impossible, but we have sufficient evidence to determine that both molecules are present in the 10.2 minute peak( the earlier section of the peak has an MS spectra consistent with the C16 alcohol, and the later section is consistent with the C17 alkene). The C13 and C15 alkane peaks showed MS spectra highly consistent with C13 and C15 alkane reference spectra(for these spectra, as well as the C17 alkene spectrum, refer to our [https://2011.igem.org/Team:Washington/Alkanes/spectra Spectra Page.]). In addition, both alkane molecules eluted off of the GC at the same time as C13 and C15 alkane standards, further increasing confidence that the 8.2 min and 9.2 min peaks do correspond to the C13 and C15 alkanes. Odd chain length alkane production is what is expected from our system, as ADC removes a carbonyl group from even chain length aldehydes produced by AAR, yielding an odd chain length alkane. Using the PetroBrick, we can turn simple sugars into diesel, a fuel fully compatible with modern infrastructure.<br />
<br />
<br />
----<br />
<br />
='''Initial Quantization of Alkane Production'''=<br />
In order to be able to know how much alkane was being produced by our ''E. coli'', we spiked known amounts of alkane into cell cultures known to not produce alkanes. We then extracted using ethyl acetate, and analyzed extracts using GC-MS. Since peak area corresponds to the amount of each substance present, we used these GC plots to make a standard curve that allows us to convert peak area into an absolute yield. To determine how much alkane was being produced by the Petrobrick, we grew up 3 MG1655 cell cultures transformed with the PetroBrick in M9 production media ([https://2011.igem.org/Team:Washington/alkanebiosynthesis link]), and analyzed using GC-MS.<br />
<br />
[[File:Washington_alkanestandardcurve.png|right|450px|thumb|Standard curves for converting peak area to an absolute amount. Note that these curves is almost perfectly linear. In addition, the curve generated from each alkane is nearly identical, allowing us to use 1, average curve for all 3 different alkanes.]]<br />
[[File:Washinton 2011 Pre-Optimization Quant.png|left|450px|thumb|Diagram showing yields of the C13 and C15 alkanes. Note: The C17 alkene is not included due to inability to quantify.]]<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
='''Optimized Production'''=<br />
By optimizing the method of growth, media, vectors, and cell line we were able to successfully increase alkane yield over 80-fold. Note that this improvement doesn't include C17 alkene production, as we were unable to quantify the C17 alkene peak due to co-elution with the C14 alcohol. The parameters we adjusted for optimization are discussed in detail at [https://2011.igem.org/Team:Washington/Alkanes/Future/Vector our systems optimization] page under future directions, as this is an on going process.<br />
<br />
[[File:Washinton 2011 Optimization Quant.png|center|550px|thumb|Our optimized growth conditions resulted in an 80 fold increase in total alkane yield.]]<br />
='''The FabBrick, a module for even chain length alkane production'''=<br />
In order to more closely match the composition of diesel, we wanted a way to make the C14 and C16 alkanes. We found an enzyme, FabH2 cfrom ''Bacillus subtilis'' that has been hypothesized to start fatty acid biosynthesis with a 3 carbon unit instead of the normal 2 carbon unit. We thought that if we could express FabH2 together with the PetroBrick, we could make even chain length alkanes. WE cloned FabH2 into a alc inducible promoter to form [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590064 the FabBrick], an add-on module to teh PetroBrick system. The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/PetroBrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When the FabBrick is induced, we see a new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that of alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinantly, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels. The FabBrick system shows the modularity and expandability of the PetroBrick system, and is a first proof of concept of the expansion of PetroBricks to produce a wider range of products. For more information on FabH2, refer to our[https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2 future directions page ].<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/ResultsTeam:Washington/Alkanes/Results2011-10-29T00:32:30Z<p>Hargem: /* The FabBrick, a module for even chain length alkane production */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
<center><big><big><big><big>'''Diesel Production: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
='''GCMS confirms The PetroBrick enables diesel production from sugar using ''E. coli'''''=<br />
<br />
We performed GC-MS analysis on extracts from cell cultures expressing both [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 Acyl-ACP Reductase] (AAR) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 Aldehyde Decarbonylase] (ADC) (this composite part is designated [http://partsregistry.org/Part:BBa_K590025 The PetroBrick]). To act as controls and in order to show that the alkane production system is working as expected, we also analyzed cell cultures expressing either only AAR, or only ADC.<br />
<br />
<br />
[[File:Washington PetroBrick.png|300px|frameless|border|left|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
[[Image:Washinton_AARADCGC-MS.png|center|600px|thumb|GCMS confirms PetroBrick diesel production from sugar using E. coli]]<br />
<br />
<br><br />
<br />
'''No significant hydrocarbons peaks in samples extracted from cells expressing only ADC.''' The blue GCMS chromatagram trace on the bottom show no significant peaks are found on the ADC only control within the time range that we expect alkane signal based upon GC-MS runs on chemical standards (8-10.5 minutes). This is expected, as ''E. coli'' does not normally produce any long chain length aldehyde substrates.<br />
<br />
'''C16 alcohols are observed when AAR is expressed on its own.''' The red GCMS chromatagram trace in the middle has a significant peak at 10.2 minutes corresponding to the C16 alcohol (as confirmed by comparison of the peak's MS spectra to a reference library) is observed when AAR is expressed in MG1655 E. coli. The production of C16 alcohols in cells expressing AAR (but not in cells expressing only ADC) is consistent with AAR reducing even chain length Acyl-ACPs into even chain length fatty aldehydes, which are further reduced by aldehyde dehydrogenases to the alcohol.<br />
<br />
'''The PetroBrick Works! C13, C15, and C17 alkanes are produced when both AAR and ADC are expressed.''' <br />
[[File:Washington2011 Spectra.png|right|550px|thumb|Comparasion of the NIST reference C15 alkane spectrum( in blue) to the C15 peak spectrum taken from a PetroBrick culture (in red). Note the strong m/z peak at 212, corresponding to the parent ion of the C15 alkane. For spectra of the C13 alkane and C17 alkene, refer to our [https://2011.igem.org/Team:Washington/Alkanes/spectra Spectra Page.]]]<br />
The green GCMS chromatagram trace in the middle has a significant new peaks corresponding to the C13 (8.2min) and C15 (9.2min) alkanes, as well as a peak at 10.2 minutes that contains both the C14 alcohol and the C17 alkene. The fact that the C17 alkene peak overlaps with the C14 alcohol peak makes exact quantification of C17 akene yields impossible, but we have sufficient evidence to determine that both molecules are present in the 10.2 minute peak( the earlier section of the peak has an MS spectra consistent with the C16 alcohol, and the later section is consistent with the C17 alkene). The C13 and C15 alkane peaks showed MS spectra highly consistent with C13 and C15 alkane reference spectra(for these spectra, as well as the C17 alkene spectrum, refer to our [https://2011.igem.org/Team:Washington/Alkanes/spectra Spectra Page.]). In addition, both alkane molecules eluted off of the GC at the same time as C13 and C15 alkane standards, further increasing confidence that the 8.2 min and 9.2 min peaks do correspond to the C13 and C15 alkanes. Odd chain length alkane production is what is expected from our system, as ADC removes a carbonyl group from even chain length aldehydes produced by AAR, yielding an odd chain length alkane. Using the PetroBrick, we can turn simple sugars into diesel, a fuel fully compatible with modern infrastructure.<br />
<br />
<br />
----<br />
<br />
='''Initial Quantization of Alkane Production'''=<br />
In order to be able to know how much alkane was being produced by our ''E. coli'', we spiked known amounts of alkane into cell cultures known to not produce alkanes. We then extracted using ethyl acetate, and analyzed extracts using GC-MS. Since peak area corresponds to the amount of each substance present, we used these GC plots to make a standard curve that allows us to convert peak area into an absolute yield. To determine how much alkane was being produced by the Petrobrick, we grew up 3 MG1655 cell cultures transformed with the PetroBrick in M9 production media ([https://2011.igem.org/Team:Washington/alkanebiosynthesis link]), and analyzed using GC-MS.<br />
<br />
[[File:Washington_alkanestandardcurve.png|right|450px|thumb|Standard curves for converting peak area to an absolute amount. Note that these curves is almost perfectly linear. In addition, the curve generated from each alkane is nearly identical, allowing us to use 1, average curve for all 3 different alkanes.]]<br />
[[File:Washinton 2011 Pre-Optimization Quant.png|left|450px|thumb|Diagram showing yields of the C13 and C15 alkanes. Note: The C17 alkene is not included due to inability to quantify.]]<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
='''Optimized Production'''=<br />
By optimizing the method of growth, media, vectors, and cell line we were able to successfully increase alkane yield over 80-fold. Note that this improvement doesn't include C17 alkene production, as we were unable to quantify the C17 alkene peak due to co-elution with the C14 alcohol. The parameters we adjusted for optimization are discussed in detail at [https://2011.igem.org/Team:Washington/Alkanes/Future/Vector our systems optimization] page under future directions, as this is an on going process.<br />
<br />
[[File:Washinton 2011 Optimization Quant.png|center|550px|thumb|Our optimized growth conditions resulted in an 80 fold increase in total alkane yield.]]<br />
='''The FabBrick, a module for even chain length alkane production'''=<br />
In order to more closely match the composition of diesel, we wanted a way to make the C14 and C16 alkanes. We found an enzyme, FabH2 cfrom ''Bacillus subtilis'' that has been hypothesized to start fatty acid biosynthesis with a 3 carbon unit instead of the normal 2 carbon unit. We thought that if we could express FabH2 together with the PetroBrick, we could make even chain length alkanes. WE cloned FabH2 into a alc inducible promoter to form [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590064 the FabBrick], an add-on module to teh PetroBrick system. The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/PetroBrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When the FabBrick is induced, we see a new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that of alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinantly, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels. The FabBrick system shows the modularity and expandability of the PetroBrick system, and is a first proof of concept of the expansion of PetroBricks to produce a wider range of products. For more information on FabH2, refer to our[[https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2 future directions page ]].<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/ResultsTeam:Washington/Alkanes/Results2011-10-29T00:32:11Z<p>Hargem: /* The FabBrick, a module for even chain length alkane production */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
<center><big><big><big><big>'''Diesel Production: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
='''GCMS confirms The PetroBrick enables diesel production from sugar using ''E. coli'''''=<br />
<br />
We performed GC-MS analysis on extracts from cell cultures expressing both [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 Acyl-ACP Reductase] (AAR) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 Aldehyde Decarbonylase] (ADC) (this composite part is designated [http://partsregistry.org/Part:BBa_K590025 The PetroBrick]). To act as controls and in order to show that the alkane production system is working as expected, we also analyzed cell cultures expressing either only AAR, or only ADC.<br />
<br />
<br />
[[File:Washington PetroBrick.png|300px|frameless|border|left|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
[[Image:Washinton_AARADCGC-MS.png|center|600px|thumb|GCMS confirms PetroBrick diesel production from sugar using E. coli]]<br />
<br />
<br><br />
<br />
'''No significant hydrocarbons peaks in samples extracted from cells expressing only ADC.''' The blue GCMS chromatagram trace on the bottom show no significant peaks are found on the ADC only control within the time range that we expect alkane signal based upon GC-MS runs on chemical standards (8-10.5 minutes). This is expected, as ''E. coli'' does not normally produce any long chain length aldehyde substrates.<br />
<br />
'''C16 alcohols are observed when AAR is expressed on its own.''' The red GCMS chromatagram trace in the middle has a significant peak at 10.2 minutes corresponding to the C16 alcohol (as confirmed by comparison of the peak's MS spectra to a reference library) is observed when AAR is expressed in MG1655 E. coli. The production of C16 alcohols in cells expressing AAR (but not in cells expressing only ADC) is consistent with AAR reducing even chain length Acyl-ACPs into even chain length fatty aldehydes, which are further reduced by aldehyde dehydrogenases to the alcohol.<br />
<br />
'''The PetroBrick Works! C13, C15, and C17 alkanes are produced when both AAR and ADC are expressed.''' <br />
[[File:Washington2011 Spectra.png|right|550px|thumb|Comparasion of the NIST reference C15 alkane spectrum( in blue) to the C15 peak spectrum taken from a PetroBrick culture (in red). Note the strong m/z peak at 212, corresponding to the parent ion of the C15 alkane. For spectra of the C13 alkane and C17 alkene, refer to our [https://2011.igem.org/Team:Washington/Alkanes/spectra Spectra Page.]]]<br />
The green GCMS chromatagram trace in the middle has a significant new peaks corresponding to the C13 (8.2min) and C15 (9.2min) alkanes, as well as a peak at 10.2 minutes that contains both the C14 alcohol and the C17 alkene. The fact that the C17 alkene peak overlaps with the C14 alcohol peak makes exact quantification of C17 akene yields impossible, but we have sufficient evidence to determine that both molecules are present in the 10.2 minute peak( the earlier section of the peak has an MS spectra consistent with the C16 alcohol, and the later section is consistent with the C17 alkene). The C13 and C15 alkane peaks showed MS spectra highly consistent with C13 and C15 alkane reference spectra(for these spectra, as well as the C17 alkene spectrum, refer to our [https://2011.igem.org/Team:Washington/Alkanes/spectra Spectra Page.]). In addition, both alkane molecules eluted off of the GC at the same time as C13 and C15 alkane standards, further increasing confidence that the 8.2 min and 9.2 min peaks do correspond to the C13 and C15 alkanes. Odd chain length alkane production is what is expected from our system, as ADC removes a carbonyl group from even chain length aldehydes produced by AAR, yielding an odd chain length alkane. Using the PetroBrick, we can turn simple sugars into diesel, a fuel fully compatible with modern infrastructure.<br />
<br />
<br />
----<br />
<br />
='''Initial Quantization of Alkane Production'''=<br />
In order to be able to know how much alkane was being produced by our ''E. coli'', we spiked known amounts of alkane into cell cultures known to not produce alkanes. We then extracted using ethyl acetate, and analyzed extracts using GC-MS. Since peak area corresponds to the amount of each substance present, we used these GC plots to make a standard curve that allows us to convert peak area into an absolute yield. To determine how much alkane was being produced by the Petrobrick, we grew up 3 MG1655 cell cultures transformed with the PetroBrick in M9 production media ([https://2011.igem.org/Team:Washington/alkanebiosynthesis link]), and analyzed using GC-MS.<br />
<br />
[[File:Washington_alkanestandardcurve.png|right|450px|thumb|Standard curves for converting peak area to an absolute amount. Note that these curves is almost perfectly linear. In addition, the curve generated from each alkane is nearly identical, allowing us to use 1, average curve for all 3 different alkanes.]]<br />
[[File:Washinton 2011 Pre-Optimization Quant.png|left|450px|thumb|Diagram showing yields of the C13 and C15 alkanes. Note: The C17 alkene is not included due to inability to quantify.]]<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
='''Optimized Production'''=<br />
By optimizing the method of growth, media, vectors, and cell line we were able to successfully increase alkane yield over 80-fold. Note that this improvement doesn't include C17 alkene production, as we were unable to quantify the C17 alkene peak due to co-elution with the C14 alcohol. The parameters we adjusted for optimization are discussed in detail at [https://2011.igem.org/Team:Washington/Alkanes/Future/Vector our systems optimization] page under future directions, as this is an on going process.<br />
<br />
[[File:Washinton 2011 Optimization Quant.png|center|550px|thumb|Our optimized growth conditions resulted in an 80 fold increase in total alkane yield.]]<br />
='''The FabBrick, a module for even chain length alkane production'''=<br />
In order to more closely match the composition of diesel, we wanted a way to make the C14 and C16 alkanes. We found an enzyme, FabH2 cfrom ''Bacillus subtilis'' that has been hypothesized to start fatty acid biosynthesis with a 3 carbon unit instead of the normal 2 carbon unit. We thought that if we could express FabH2 together with the PetroBrick, we could make even chain length alkanes. WE cloned FabH2 into a alc inducible promoter to form [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590064 the FabBrick], an add-on module to teh PetroBrick system. The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/PetroBrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When the FabBrick is induced, we see a new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that of alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinantly, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels. The FabBrick system shows the modularity and expandability of the PetroBrick system, and is a first proof of concept of the expansion of PetroBricks to produce a wider range of products. For more information on FabH2, refer to our[[ future directions page https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2]].<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/ResultsTeam:Washington/Alkanes/Results2011-10-29T00:31:51Z<p>Hargem: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
<center><big><big><big><big>'''Diesel Production: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
='''GCMS confirms The PetroBrick enables diesel production from sugar using ''E. coli'''''=<br />
<br />
We performed GC-MS analysis on extracts from cell cultures expressing both [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 Acyl-ACP Reductase] (AAR) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 Aldehyde Decarbonylase] (ADC) (this composite part is designated [http://partsregistry.org/Part:BBa_K590025 The PetroBrick]). To act as controls and in order to show that the alkane production system is working as expected, we also analyzed cell cultures expressing either only AAR, or only ADC.<br />
<br />
<br />
[[File:Washington PetroBrick.png|300px|frameless|border|left|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
[[Image:Washinton_AARADCGC-MS.png|center|600px|thumb|GCMS confirms PetroBrick diesel production from sugar using E. coli]]<br />
<br />
<br><br />
<br />
'''No significant hydrocarbons peaks in samples extracted from cells expressing only ADC.''' The blue GCMS chromatagram trace on the bottom show no significant peaks are found on the ADC only control within the time range that we expect alkane signal based upon GC-MS runs on chemical standards (8-10.5 minutes). This is expected, as ''E. coli'' does not normally produce any long chain length aldehyde substrates.<br />
<br />
'''C16 alcohols are observed when AAR is expressed on its own.''' The red GCMS chromatagram trace in the middle has a significant peak at 10.2 minutes corresponding to the C16 alcohol (as confirmed by comparison of the peak's MS spectra to a reference library) is observed when AAR is expressed in MG1655 E. coli. The production of C16 alcohols in cells expressing AAR (but not in cells expressing only ADC) is consistent with AAR reducing even chain length Acyl-ACPs into even chain length fatty aldehydes, which are further reduced by aldehyde dehydrogenases to the alcohol.<br />
<br />
'''The PetroBrick Works! C13, C15, and C17 alkanes are produced when both AAR and ADC are expressed.''' <br />
[[File:Washington2011 Spectra.png|right|550px|thumb|Comparasion of the NIST reference C15 alkane spectrum( in blue) to the C15 peak spectrum taken from a PetroBrick culture (in red). Note the strong m/z peak at 212, corresponding to the parent ion of the C15 alkane. For spectra of the C13 alkane and C17 alkene, refer to our [https://2011.igem.org/Team:Washington/Alkanes/spectra Spectra Page.]]]<br />
The green GCMS chromatagram trace in the middle has a significant new peaks corresponding to the C13 (8.2min) and C15 (9.2min) alkanes, as well as a peak at 10.2 minutes that contains both the C14 alcohol and the C17 alkene. The fact that the C17 alkene peak overlaps with the C14 alcohol peak makes exact quantification of C17 akene yields impossible, but we have sufficient evidence to determine that both molecules are present in the 10.2 minute peak( the earlier section of the peak has an MS spectra consistent with the C16 alcohol, and the later section is consistent with the C17 alkene). The C13 and C15 alkane peaks showed MS spectra highly consistent with C13 and C15 alkane reference spectra(for these spectra, as well as the C17 alkene spectrum, refer to our [https://2011.igem.org/Team:Washington/Alkanes/spectra Spectra Page.]). In addition, both alkane molecules eluted off of the GC at the same time as C13 and C15 alkane standards, further increasing confidence that the 8.2 min and 9.2 min peaks do correspond to the C13 and C15 alkanes. Odd chain length alkane production is what is expected from our system, as ADC removes a carbonyl group from even chain length aldehydes produced by AAR, yielding an odd chain length alkane. Using the PetroBrick, we can turn simple sugars into diesel, a fuel fully compatible with modern infrastructure.<br />
<br />
<br />
----<br />
<br />
='''Initial Quantization of Alkane Production'''=<br />
In order to be able to know how much alkane was being produced by our ''E. coli'', we spiked known amounts of alkane into cell cultures known to not produce alkanes. We then extracted using ethyl acetate, and analyzed extracts using GC-MS. Since peak area corresponds to the amount of each substance present, we used these GC plots to make a standard curve that allows us to convert peak area into an absolute yield. To determine how much alkane was being produced by the Petrobrick, we grew up 3 MG1655 cell cultures transformed with the PetroBrick in M9 production media ([https://2011.igem.org/Team:Washington/alkanebiosynthesis link]), and analyzed using GC-MS.<br />
<br />
[[File:Washington_alkanestandardcurve.png|right|450px|thumb|Standard curves for converting peak area to an absolute amount. Note that these curves is almost perfectly linear. In addition, the curve generated from each alkane is nearly identical, allowing us to use 1, average curve for all 3 different alkanes.]]<br />
[[File:Washinton 2011 Pre-Optimization Quant.png|left|450px|thumb|Diagram showing yields of the C13 and C15 alkanes. Note: The C17 alkene is not included due to inability to quantify.]]<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
='''Optimized Production'''=<br />
By optimizing the method of growth, media, vectors, and cell line we were able to successfully increase alkane yield over 80-fold. Note that this improvement doesn't include C17 alkene production, as we were unable to quantify the C17 alkene peak due to co-elution with the C14 alcohol. The parameters we adjusted for optimization are discussed in detail at [https://2011.igem.org/Team:Washington/Alkanes/Future/Vector our systems optimization] page under future directions, as this is an on going process.<br />
<br />
[[File:Washinton 2011 Optimization Quant.png|center|550px|thumb|Our optimized growth conditions resulted in an 80 fold increase in total alkane yield.]]<br />
='''The FabBrick, a module for even chain length alkane production'''=<br />
In order to more closely match the composition of diesel, we wanted a way to make the C14 and C16 alkanes. We found an enzyme, FabH2 cfrom ''Bacillus subtilis'' that has been hypothesized to start fatty acid biosynthesis with a 3 carbon unit instead of the normal 2 carbon unit. We thought that if we could express FabH2 together with the PetroBrick, we could make even chain length alkanes. WE cloned FabH2 into a alc inducible promoter to form [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590064 the FabBrick], an add-on module to teh PetroBrick system. The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/PetroBrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When the FabBrick is induced, we see a new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that of alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinantly, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels. The FabBrick system shows the modularity and expandability of the PetroBrick system, and is a first proof of concept of the expansion of PetroBricks to produce a wider range of products. For more information on FabH2, refer to our[ future directions page https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2].<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/FutureTeam:Washington/Alkanes/Future2011-10-29T00:30:53Z<p>Hargem: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Future Directions'''</big></big></big></big></center><br><br><br />
<br />
Our current ''in vivo'' alkane production system efficiently makes C15 alkanes. However, to be efficient enough for factory production, there are two broad goals to be done: <br />
# Increase production efficiency <br />
# Increase the diversity of the range of alkanes that can be produced. <br />
# Increase the scale of the system for industrial processes. <br />
We have already begun efforts to expand the efficiency and scope of alkane production, '''check them out below!'''<br />
<br />
[[Image:UW_2011_Alkane_Future_Work_Image.png|7px||right]]<br />
<br />
[[Image:Washington system optimization.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Vector]]<br />
<br><br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Vector '''System Optimization''']<br />
:<nowiki> The efficiency of this system was reported to be much higher than our initial system, but the growth conditions of the assay and the DNA was not available to us. We increased production efficiency by altering the initial environmental conditions in the assay.</nowiki><br />
<br><br><br />
<br />
[[Image:Washington_2011_ADC_Redesign.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/DecarbDesign]]<br />
<br><br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/DecarbDesign '''Decarbonylase Redesign''']<br />
:<nowiki>One way to diversify the kind of alkanes produced is to alter the substrate specificity of the proteins involved. Since an alternative alkane-producing enzyme has not been found, we decided to mutate the aldehyde decarbonylase to produce shorter-chain alkanes.</nowiki><br />
<br><br><br />
<br />
[[Image:Washington_2011_LuxCDE.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/LuxCDE]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/LuxCDE '''Alternative Aldehyde''']<br />
:<nowiki>Another way to diversify our system is to use alternative proteins. Our current system uses acyl-ACP reductase, and we've identified an hypothetical alternative system that produces aldehydes: LuxCDE, made from parts from the 2010 competition.</nowiki><br />
<br><br><br><br />
<br />
[[Image:Washington_2011_fabh2_branch.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2 '''Alternative Alkane Products ''']<br />
:<nowiki> Our system is only capable of producing unbranched, odd chain length alkanes, as the cell mainly utilizes straight chained, even chain length fatty acids. However, fuel we use is composed of a wide range of products. By changing which fatty acids are made by alkane producing cells, we could theoretically change which alkanes are being produced by our system.<br />
<br><br />
<br />
[[Image:Washington 2011 Protein Localization.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Localization]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Localization '''Enzyme Localization''']<br />
:<nowiki> The easiest way to increase yield is to increase the concentration; if the number of molecules remained the same but the volume decreased, the reaction speed increases with occurrences of molecular collisions. However, this is only easily done with purified protein assays. However, there are ways to "increase the concentration" of reactions in cells. We cannot make the cells literally denser, but we can localize the enzymes involved in the reaction, which either decreases the volume of the cell in which the enzymes reside and/or limit the number of reaction steps.</nowiki><br />
<br />
[[Image:Washington_2011_Alternate_Chassis.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Yeast]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Yeast '''Alternative Chassis''']<br />
:<nowiki> By synthesizing alkanes in organisms, alkanes can become a useful renewable energy source; CO2 emitted from burning the alkanes are converted to glucose through photosynthesis, and then the glucose can be fed to alkane-synthesizing organisms to restart the cycle. This cycle can be drastically improved if there were fewer steps in the cycle. We explored the possibility of optimizing the alkane biosynthesis system in different micro-organisms, with the idea that perhaps with autotrophic organisms, we do not need to obtain glucose for the reaction.</nowiki><br />
<br />
<br />
<br />
[[Image:Washington_FB.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Modeling]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Modeling '''System Modeling''']<br />
:<nowiki> We have begun Flux Balance Modeling the alkane production system in order to better understand the carbon flux through the pathway. We believe that by understanding the curent flux through the pathway we can intelligently reengineer the platform in order to maximize flux to alkanes. </nowiki></div>Hargemhttp://2011.igem.org/Team:Washington/Team/MembersTeam:Washington/Team/Members2011-10-28T06:04:40Z<p>Hargem: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------><br />
<br />
<center><big><big><big><big>'''Who we are'''</big></big></big></big></center><br><br />
<center><br />
<gallery caption="Undergraduate Team Members" widths="180px" heights="120px" perrow="4"><br />
Image:Washington Casey Ager Profile1.jpg|<center>Casey Ager <br/> Biochemistry</center><br />
Image:Washington Photo-0088A.png|<center>Juhye An <br/> Biochemistry </center><br />
Image: Profile_Michael_Brasino.jpg|<center>Michael Brasino <br/> Materials Science and Engineering</center><br />
Image:Washington MC profilepic1.jpg|<center>Marika Cheng <br/> Microbiology</center><br />
Image:Washington_choe_chris.png|<center>Chris Choe <br/> Undecided</center><br />
Image:Washington DeLeon 2011.jpg|<center>Justin De Leon <br/> Microbiology</center><br />
File:Princess Peachy.JPG|<center>Sydney Gordon <br/> Biochemistry, Music</center><br />
Image:CIMG0021.jpg|<center>Daniel Hadidi <br/> Neurobiology</center><br />
Image:Washington_Matthew_Harger.JPG|<center>Matthew Harger <br/> Cellular and Molecular Biology</center><br />
Image:Washington_Elaine.jpg|<center>Elaine Lai <br/> Microbiology, Chemistry</center><br />
Image:Washington_2011_Lab_Ben.png|<center>Benjamin Mo <br/> Bioengineering</center><br />
Image:Washington 2011 CIMG0015-A.jpg|<center>Austin Moon <br/> Molecular Biology</center><br />
Image:Washington_iGEM2011_Rashmi.jpg|<center>Rashmi Ravichandran <br/> Microbiology</center><br />
Image:rsz_cats.png|<center>Seth Sagulo <br/> Biochemistry</center><br />
File:Washington_Liz.png|<center>Liz Stanley <br/> Microbiology, Chemistry</center><br />
Image:Washington_Angus_Pic.jpg|<center>Angus Toland <br/> Microbiology</center><br />
Image:Washington_Sarah.jpg|<center>Sarah Wolf <br/> Biochemistry</center><br />
Image:Washington_igem11whowearepicture.jpg|<center>SauShun (Alicia) Wong <br/> Materials Science and Engineering</center><br />
Image:Washington_Cindy.jpg|<center>Cindy Wu <br/> Cellular and Molecular Biology</center><br />
Image:Washington_Sean_Wu.jpg|<center>Sean Wu <br/> Computer Science and Engineering</center><br />
File:Washington_EmilyYang.jpg|<center>Emily Yang <br/> Bioengineering</center><br />
Image:Washington_lei_zheg.png|<center>Lei Zheng <br/> Biochemistry</center><br />
Image:Washington_david_zong.jpg|<center>David Zong <br/> Bioengineering</center><br />
<br />
</gallery><br />
<br />
<br />
<gallery caption="Advisors" widths="180px" heights="120px" perrow="4"><br />
File:Aaron_bike_UW.png|<center>Aaron Chevalier <br/> Bioengineering </center><br />
Image:Regbert.jpg|<center>Rob Egbert <br/> Electrical Engineering </center><br />
Image:CEiben.jpg|<center>Chris Eiben <br/> Biochemistry </center><br />
Image:IMG_0129.jpg|<center>Jeremy Mills <br/> Biochemistry </center><br />
Image:Siegel_UW_2011.jpg|<center>Justin Siegel <br/> Biomolecular Structure and Design</center><br />
Image:Washington2011_Matthew-d-smith.jpg|<center>Matt Smith <br/> Molecular and Cellular Biology</center><br />
File:Washington_Ingrid_Pic_1.jpg|<center>Ingrid Swanson Pultz<br/> Microbiology </center><br />
</gallery><br />
<br />
<br />
<gallery caption="Faculty" widths="180px" heights="120px" perrow="5"><br />
image:Washington_David_Baker.jpg|<center>David Baker <br/> Biochemistry</center><br />
Image:Washington_Klavins.jpg|<center>Eric Klavins <br/> Electrical Engineering</center><br />
File:Washington_Sauro.png|<center>Herbert Sauro <br/>Bioengineering</center><br />
</gallery><br />
<br />
<br />
<gallery caption="Collaborators and Support" widths="140px" heights="100px" perrow="5"><br />
<br />
Image:Washington_OSLI.png|<center><b>Oil Sands Leadership Initiative</b><br/>Funding to support Travel and Registration Costs</center><br />
<br />
Image:Washington_UniversitySeal.gif|<center><b>UW Biochemistry</b><br/>Lab space</center><br />
<br />
Image:Washington_Anaspec.gif|<center><b>Anaspec</b><br/>Peptide Discounts</center><br />
<br />
Image:Washington_ARPA-E_Logo.png|<center><b>Advanced Research Projects Agency - Energy</b><br/>Registration Support</center><br />
<br />
Image:Washington2011_Hhmi_362_72.jpg|<center><b>Howard Hughes Medical Institute</b><br/>Lab materials and supplies</center><br />
<br />
<br />
</gallery><br />
</center><br />
<br><br />
== '''Who did what''' ==<br />
<br />
The teams were assembled during our winter quarter. During this term, we went around to classes and had weekly meetings to introduce students to synthetic biology through a series of guest lectures. During the spring, students participated in brainstorming projects related to the sponsoring Faculty advisors' labs (Baker and Klavins) and came up with the project they wanted to carry out. Students also planned and participated in community outreach events where we taught the local public about synthetic biology. Over the summer, students completed all of the experimental work, but worked closely with both graduate students and faculty advisors to plan the most pertinent experiments and make the most of our limited time. <br />
<br />
'''The work documented here in this wiki and on our presentation is entirely the work of iGEM students - it does not represent the project of any host lab, advisor, or instructor participating in iGEM.'''<br />
<br />
=== Diesel Production ===<br />
<br />
After producing promising results, in future directions:<br />
<br />
Casey Ager, Austin Moon, and Seth Sagulo worked on Enzyme Localization via Direct Fusion and Zinc Finger Fusion methods.<br />
<br />
Juhye An, Elaine Lai, and Benjamin Mo worked on Decarbonylase Redesign<br />
<br />
Marika Cheng and Justin De Leon worked on Alternative Chassis<br />
<br />
Chris Choe and David Zong worked on Alternate Aldehyde Production<br />
<br />
Matthew Harger worked on Branched Alkanes Production<br />
<br />
Matthew Harger and Lei Zheng worked on System Optimization<br />
<br />
Emily Yang worked on modelling the alkane production pathway. <br />
<br />
=== Gluten Destruction ===<br />
<br />
Sydney Gordon, Daniel Hadidi, Liz Stanley, Angus Toland, Sarah Wolf, and Sean Wu designed, built, and tested Kumamolisin-As and over 100 mutants to combat gluten intolerance by increasing the activity on the PQLP antigenic peptide.<br />
<br />
=== iGEM Toolkits ===<br />
<br />
Michael Brasino, Rashmi Ravichandran, and Alicia Wong worked on extracting and BioBricking the essential genes for magnetosomes, made the fusion proteins, and did the experimental measurements. They also expanded on work that was started by UW iGEM students in 2010, by constructing more Gibson-friendly plasmid backbones and characterizing the Gibson-assembly efficiencies of pSB and pGA plasmids.<br />
<br />
=== Community Outreach ===<br />
<br />
Cindy Wu headed and organized all iGEM 2011 Community Outreach events.</div>Hargemhttp://2011.igem.org/File:Washington_Sauro.pngFile:Washington Sauro.png2011-10-28T06:03:34Z<p>Hargem: </p>
<hr />
<div></div>Hargemhttp://2011.igem.org/Team:Washington/Team/MembersTeam:Washington/Team/Members2011-10-28T05:58:20Z<p>Hargem: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------><br />
<br />
<center><big><big><big><big>'''Who we are'''</big></big></big></big></center><br><br />
<center><br />
<gallery caption="Undergraduate Team Members" widths="180px" heights="120px" perrow="4"><br />
Image:Washington Casey Ager Profile1.jpg|<center>Casey Ager <br/> Biochemistry</center><br />
Image:Washington Photo-0088A.png|<center>Juhye An <br/> Biochemistry </center><br />
Image: Profile_Michael_Brasino.jpg|<center>Michael Brasino <br/> Materials Science and Engineering</center><br />
Image:Washington MC profilepic1.jpg|<center>Marika Cheng <br/> Microbiology</center><br />
Image:Washington_choe_chris.png|<center>Chris Choe <br/> Undecided</center><br />
Image:Washington DeLeon 2011.jpg|<center>Justin De Leon <br/> Microbiology</center><br />
File:Princess Peachy.JPG|<center>Sydney Gordon <br/> Biochemistry, Music</center><br />
Image:CIMG0021.jpg|<center>Daniel Hadidi <br/> Neurobiology</center><br />
Image:Washington_Matthew_Harger.JPG|<center>Matthew Harger <br/> Cellular and Molecular Biology</center><br />
Image:Washington_Elaine.jpg|<center>Elaine Lai <br/> Microbiology, Chemistry</center><br />
Image:Washington_2011_Lab_Ben.png|<center>Benjamin Mo <br/> Bioengineering</center><br />
Image:Washington 2011 CIMG0015-A.jpg|<center>Austin Moon <br/> Molecular Biology</center><br />
Image:Washington_iGEM2011_Rashmi.jpg|<center>Rashmi Ravichandran <br/> Microbiology</center><br />
Image:rsz_cats.png|<center>Seth Sagulo <br/> Biochemistry</center><br />
File:Washington_Liz.png|<center>Liz Stanley <br/> Microbiology, Chemistry</center><br />
Image:Washington_Angus_Pic.jpg|<center>Angus Toland <br/> Microbiology</center><br />
Image:Washington_Sarah.jpg|<center>Sarah Wolf <br/> Biochemistry</center><br />
Image:Washington_igem11whowearepicture.jpg|<center>SauShun (Alicia) Wong <br/> Materials Science and Engineering</center><br />
Image:Washington_Cindy.jpg|<center>Cindy Wu <br/> Cellular and Molecular Biology</center><br />
Image:Washington_Sean_Wu.jpg|<center>Sean Wu <br/> Computer Science and Engineering</center><br />
File:Washington_EmilyYang.jpg|<center>Emily Yang <br/> Bioengineering</center><br />
Image:Washington_lei_zheg.png|<center>Lei Zheng <br/> Biochemistry</center><br />
Image:Washington_david_zong.jpg|<center>David Zong <br/> Bioengineering</center><br />
<br />
</gallery><br />
<br />
<br />
<gallery caption="Advisors" widths="180px" heights="120px" perrow="4"><br />
File:Aaron_bike_UW.png|<center>Aaron Chevalier <br/> Bioengineering </center><br />
Image:Regbert.jpg|<center>Rob Egbert <br/> Electrical Engineering </center><br />
Image:CEiben.jpg|<center>Chris Eiben <br/> Biochemistry </center><br />
Image:IMG_0129.jpg|<center>Jeremy Mills <br/> Biochemistry </center><br />
Image:Siegel_UW_2011.jpg|<center>Justin Siegel <br/> Biomolecular Structure and Design</center><br />
Image:Washington2011_Matthew-d-smith.jpg|<center>Matt Smith <br/> Molecular and Cellular Biology</center><br />
File:Washington_Ingrid_Pic_1.jpg|<center>Ingrid Swanson Pultz<br/> Microbiology </center><br />
</gallery><br />
<br />
<br />
<gallery caption="Faculty" widths="180px" heights="120px" perrow="5"><br />
image:Washington_David_Baker.jpg|<center>David Baker <br/> Biochemistry</center><br />
Image:Washington_Klavins.jpg|<center>Eric Klavins <br/> Electrical Engineering</center><br />
</gallery><br />
<br />
<br />
<gallery caption="Collaborators and Support" widths="140px" heights="100px" perrow="5"><br />
<br />
Image:Washington_OSLI.png|<center><b>Oil Sands Leadership Initiative</b><br/>Funding to support Travel and Registration Costs</center><br />
<br />
Image:Washington_UniversitySeal.gif|<center><b>UW Biochemistry</b><br/>Lab space</center><br />
<br />
Image:Washington_Anaspec.gif|<center><b>Anaspec</b><br/>Peptide Discounts</center><br />
<br />
Image:Washington_ARPA-E_Logo.png|<center><b>Advanced Research Projects Agency - Energy</b><br/>Registration Support</center><br />
<br />
Image:Washington2011_Hhmi_362_72.jpg|<center><b>Howard Hughes Medical Institute</b><br/>Lab materials and supplies</center><br />
<br />
<br />
</gallery><br />
</center><br />
<br><br />
== '''Who did what''' ==<br />
<br />
The teams were assembled during our winter quarter. During this term, we went around to classes and had weekly meetings to introduce students to synthetic biology through a series of guest lectures. During the spring, students participated in brainstorming projects related to the sponsoring Faculty advisors' labs (Baker and Klavins) and came up with the project they wanted to carry out. Students also planned and participated in community outreach events where we taught the local public about synthetic biology. Over the summer, students completed all of the experimental work, but worked closely with both graduate students and faculty advisors to plan the most pertinent experiments and make the most of our limited time. <br />
<br />
'''The work documented here in this wiki and on our presentation is entirely the work of iGEM students - it does not represent the project of any host lab, advisor, or instructor participating in iGEM.'''<br />
<br />
=== Diesel Production ===<br />
<br />
After producing promising results, in future directions:<br />
<br />
Casey Ager, Austin Moon, and Seth Sagulo worked on Enzyme Localization via Direct Fusion and Zinc Finger Fusion methods.<br />
<br />
Juhye An, Elaine Lai, and Benjamin Mo worked on Decarbonylase Redesign<br />
<br />
Marika Cheng and Justin De Leon worked on Alternative Chassis<br />
<br />
Chris Choe and David Zong worked on Alternate Aldehyde Production<br />
<br />
Matthew Harger worked on Branched Alkanes Production<br />
<br />
Matthew Harger and Lei Zheng worked on System Optimization<br />
<br />
Emily Yang worked on modelling the alkane production pathway. <br />
<br />
=== Gluten Destruction ===<br />
<br />
Sydney Gordon, Daniel Hadidi, Liz Stanley, Angus Toland, Sarah Wolf, and Sean Wu designed, built, and tested Kumamolisin-As and over 100 mutants to combat gluten intolerance by increasing the activity on the PQLP antigenic peptide.<br />
<br />
=== iGEM Toolkits ===<br />
<br />
Michael Brasino, Rashmi Ravichandran, and Alicia Wong worked on extracting and BioBricking the essential genes for magnetosomes, made the fusion proteins, and did the experimental measurements. They also expanded on work that was started by UW iGEM students in 2010, by constructing more Gibson-friendly plasmid backbones and characterizing the Gibson-assembly efficiencies of pSB and pGA plasmids.<br />
<br />
=== Community Outreach ===<br />
<br />
Cindy Wu headed and organized all iGEM 2011 Community Outreach events.</div>Hargemhttp://2011.igem.org/File:Washington_EmilyYang.jpgFile:Washington EmilyYang.jpg2011-10-28T05:54:42Z<p>Hargem: </p>
<hr />
<div></div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/VectorTeam:Washington/Alkanes/Future/Vector2011-10-28T05:47:51Z<p>Hargem: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
='''<center>System Optimization</center>'''=<br />
<br />
Using our initial, non-optimized growth conditions, we were able to obtain alkane/ene yields of approximately 2 mg/L. In order to make analysis easier, and to make it easier to determine the effects of the addition of additional modules, we wanted to increase yield. Our efforts focused on how by varying system conditions, we could increase yield.<br />
<br />
=='''Sealed vs. Open Tubes'''==<br />
We were concerned that produced alkanes may have been evaporating, reducing apparent yield. Therefore, we performed tests where the tubes were either capped the standard way, or capped with foil coverting the opening, thereby reducing evaporation. Tests were conducted in glass tubes with M9 production media, with MG1655 innoculated to an initial OD600 of 1.<br />
[[File:Washinton_open_sealed.png|center|500px|thumb|Sealing Culture Tubes Increases Yield.]]<br />
Covered tubes showed significantly more alkanes, so all further tests would be conducted in sealed tubes.<br />
<br />
=='''Use of Different Strains'''==<br />
We had suspected that different strains of ''E. coli'' would produce varying amounts of alkanes. Initial experiments were done in MG1655, and we decided to test XL-1 blue (a commercial supercopentent variant of DH5a) for the ability to produce alkanes. Tests were conducted in sealed glass culture tubes in M9 production media . Cells were innoculated to an OD600 of 1 when conducting this test.<br />
[[File:Washinton_XL21vsMG1655.png|center|500px|thumb|XL-1 Blue Produces more Alkane than MG1655]]<br />
XL-1 blue was able to produce more alkanes than MG1655. Therefore, future tests were conducted in XL-1 Blue.<br />
<br />
=='''Aerobic vs Microaerobic Growth'''==<br />
In many industrial production applications, growth in conditions with little or no oxygen can improve yield. Therefore, we tested alkane yield in cultures grown in airtight vials with only a small amount of air on the top in order to severely limit oxygen availability. Tests were conducted in glass (note that the test was conducted in open culture tubes for aerobic cultures, and sealed glass vials for microaerobic cultures, with M9 production media in XL21-blue cells.)<br />
[[File:Washington_aerobicVsMicroaerobic.png|center|500px|thumb|Oxygen has a Positive Effect on Alkane Production]]<br />
Based upon this data, aerobic growth appears to be better for alkane yield. Note that this difference is not likely due to differences in growth rate, as both cultures were inoculated to a high OD600 of 1. The fact that this tube was not covered means that the actual alkane yield (before evaporation) was likely higher. Therefore, microaerobic conditions are unlikely to improve yield.<br />
<br />
=='''pSB1C3-ADC-AAR vs pSB3k3-ADC/pSB4C5-AAR'''==<br />
The original study expressed ADC and AAR on seperate low copy number vectors. If we could get high alkane yields using low copy number vectors, the lesser protien and vector production would make the use of a lower copy number vector preferable. Tests were conducted in covered glass culture tubes (aerobic conditions) with M9 production media. Cells were XL1-Blue, and were innoculated to an OD600 of 1.<br />
[[File:Washington_copynumber.png|center|500px|thumb|Greater ADC/AAR Expression Results in More Alkane Production.]]<br />
The fact that high copynumber vector use results in an increase in yield means that high expression levels of ADC and/or AAR are required for high alkane yields.<br />
<br />
=='''Media composition'''==<br />
Our initial media was based upon the media used in [[#References |[1]]]. We did not know if all of the additional components not found in a normal media (buffer, thiamine, Triton, iron) actually improved yield. In order to determine if each of these components imporved yield, we analyzed cultures inoculated into M9 medias without each of these 4 compounds. In addition, we tested media with additional trace metals. These tests were conducted in covered glass culture tubes with XL-1 blue cells.<br />
[[File:Washington_mediaconditions.png|center|500px|thumb|Effects of Media Composition on Alkane Yield.]]<br />
None of the modifications we made resulted in a significant increase in yield. Therefore, no further modifications to the media used was made.<br />
<br />
=='''Effects of Changing Initial Cell Density'''==<br />
Earlier tests were done with an initial OD600 of 1. However, we had never established what the optimal initial OD would be. If significant alkane production occurs during stationary phase, inoculation to a high initial OD may increase alkane production. For this test, we inoculated XL-1 blue (to a final OD600 in a range of 0.01 to 10) in M9 production media (in covered glass culture tubes). We extracted from these cultures after 16 hours of growth. <br />
[[File:Washington_alkaneODcurve.png|center|500px|thumb|Greater Initial Cell Density Results in Higher Alkane Yield.]]<br />
Higher cell density cultures showed significantly higher alkane yields than lower cell density cultures. The increase of alkane yields at cell densities as high as 10 implies that high amounts of alkane production occurs during stationary phase. Therefore, alkane production scales with cell density. In addition, inducing alkane production during stationary phase decreases the amount of glucose going towards cellular growth instead of alkane production.<br />
=='''Final conditions'''==<br />
Using our optimized media & growth conditions(XL-1 blue inoculated to an OD600 of 10, in M9 media, in closed glass culture tubes) , we were able to improve total alkane yield 80 fold, from approximenly 2 mg/L (after 48 hrs) to approximently 170 mg/L(after 48 hrs).<br />
[[File:Washinton 2011 Optimization Quant.png|center|550px|thumb|Our optimized growth conditions resulted in an 80 fold increase in total alkane yield.]]<br />
=='''Production Curve'''==<br />
Ideally, alkane extraction and analysis would occur after all ( or essentially all) of the glucose in the media was used up. However, we didn't know how long it would take for our cells to use up all of the glucose. In order to determine the best time for extraction, we extracted alkanes from M9 production cultures that had been producing for 6, 24, 48, and 72 hours. Three cultures were used for each timepoint.<br />
[[file:Washington_Productioncurve.png|center|550px|thumb|Total alkane yield over time under optimized conditions.]]<br />
The 24 hour timepoint was essentially identical to the 48 and 72 hour timepoints. This implies that our cells are using up 30g/L glucose sometime before 24 hours. Future work will consist of increasing glucose concentration, and increasing inoculated cell density in order to maximize alkane production per culture per day.<br />
=='''Alternate Carbon Sources'''==<br />
While previous tests were done using glucose as the sole carbon source, it would be helpful to be able to use carbon sources that can be considered waste sources. There is a large amount of interest in using non-simple sugar carbon sources, such as cellulosic biomass, and even using carbon dioxide directly. However, these carbon sources cannot be easily utilized by ''E. coli''. Glycerol is a 3-carbon triol that is a waste product in the production of current generation biodiesel(fatty acid methyl esters(FAMEs). Glycerol can be used by ''E. coli'' as a sole carbon source, so we tried to use the PetroBrick to convert glycerol into diesel. We performed standard alkane production/analysis, with the second production media being either [https://2011.igem.org/Team:Washington/alkanebiosynthesis#Current_Protocol_for_100mL_of_Media standard M9 glucose media], or M9 media varients containing an equal mass of glycerol instead of glucose, or media without any carbon source.<br />
[[file:Washington_Productioncurve.png|center|550px|thumb|Total alkane yield in M9 glucose, M9 glycerol, and M9 nedia without carbon.]]<br />
The glucose media resulted in higher total alkane yield than the glycerol media(240 mg/L vs. 56.3 mg/L), but this may be due to media conditions being optimized for glucose as a carbon source insteam od glycerol. Further optimization may be able to improve on alkane yield when using glycerol as a carbon source. In media without carbon, we observed negligable alkane production(4.7 mg/L). This indicates that alkane production is due to conversion of carbon in production media into alkanes, not due to the conversion of cellular biomass into alkanes. This means that initial growth for cell density can occur in a rich media without having any major influence on yield. <br />
=='''References'''==<br />
Microbial Biosynthesis of Alkanes Andreas Schirmer, Mathew A. Rude, Xuezhi Li, Emanuela Popova and Stephen B. del Cardayre Science 30 July 2010: Vol. 329 no. 5991 pp. 559-562 http://www.sciencemag.org/content/329/5991/559</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/VectorTeam:Washington/Alkanes/Future/Vector2011-10-28T05:44:22Z<p>Hargem: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
='''<center>System Optimization</center>'''=<br />
<br />
Using our initial, non-optimized growth conditions, we were able to obtain alkane/ene yields of approximately 2 mg/L. In order to make analysis easier, and to make it easier to determine the effects of the addition of additional modules, we wanted to increase yield. Our efforts focused on how by varying system conditions, we could increase yield.<br />
<br />
=='''Sealed vs. Open Tubes'''==<br />
We were concerned that produced alkanes may have been evaporating, reducing apparent yield. Therefore, we performed tests where the tubes were either capped the standard way, or capped with foil coverting the opening, thereby reducing evaporation. Tests were conducted in glass tubes with M9 production media, with MG1655 innoculated to an initial OD600 of 1.<br />
[[File:Washinton_open_sealed.png|center|500px|thumb|Sealing Culture Tubes Increases Yield.]]<br />
Covered tubes showed significantly more alkanes, so all further tests would be conducted in sealed tubes.<br />
<br />
=='''Use of Different Strains'''==<br />
We had suspected that different strains of ''E. coli'' would produce varying amounts of alkanes. Initial experiments were done in MG1655, and we decided to test XL-1 blue (a commercial supercopentent variant of DH5a) for the ability to produce alkanes. Tests were conducted in sealed glass culture tubes in M9 production media . Cells were innoculated to an OD600 of 1 when conducting this test.<br />
[[File:Washinton_XL21vsMG1655.png|center|500px|thumb|XL-1 Blue Produces more Alkane than MG1655]]<br />
XL-1 blue was able to produce more alkanes than MG1655. Therefore, future tests were conducted in XL-1 Blue.<br />
<br />
=='''Aerobic vs Microaerobic Growth'''==<br />
In many industrial production applications, growth in conditions with little or no oxygen can improve yield. Therefore, we tested alkane yield in cultures grown in airtight vials with only a small amount of air on the top in order to severely limit oxygen availability. Tests were conducted in glass (note that the test was conducted in open culture tubes for aerobic cultures, and sealed glass vials for microaerobic cultures, with M9 production media in XL21-blue cells.)<br />
[[File:Washington_aerobicVsMicroaerobic.png|center|500px|thumb|Oxygen has a Positive Effect on Alkane Production]]<br />
Based upon this data, aerobic growth appears to be better for alkane yield. Note that this difference is not likely due to differences in growth rate, as both cultures were inoculated to a high OD600 of 1. The fact that this tube was not covered means that the actual alkane yield (before evaporation) was likely higher. Therefore, microaerobic conditions are unlikely to improve yield.<br />
<br />
=='''pSB1C3-ADC-AAR vs pSB3k3-ADC/pSB4C5-AAR'''==<br />
The original study expressed ADC and AAR on seperate low copy number vectors. If we could get high alkane yields using low copy number vectors, the lesser protien and vector production would make the use of a lower copy number vector preferable. Tests were conducted in covered glass culture tubes (aerobic conditions) with M9 production media. Cells were XL1-Blue, and were innoculated to an OD600 of 1.<br />
[[File:Washington_copynumber.png|center|500px|thumb|Greater ADC/AAR Expression Results in More Alkane Production.]]<br />
The fact that high copynumber vector use results in an increase in yield means that high expression levels of ADC and/or AAR are required for high alkane yields.<br />
<br />
=='''Media composition'''==<br />
Our initial media was based upon the media used in [[#References |[1]]]. We did not know if all of the additional components not found in a normal media (buffer, thiamine, Triton, iron) actually improved yield. In order to determine if each of these components imporved yield, we analyzed cultures inoculated into M9 medias without each of these 4 compounds. In addition, we tested media with additional trace metals. These tests were conducted in covered glass culture tubes with XL-1 blue cells.<br />
[[File:Washington_mediaconditions.png|center|500px|thumb|Effects of Media Composition on Alkane Yield.]]<br />
None of the modifications we made resulted in a significant increase in yield. Therefore, no further modifications to the media used was made.<br />
<br />
=='''Effects of Changing Initial Cell Density'''==<br />
Earlier tests were done with an initial OD600 of 1. However, we had never established what the optimal initial OD would be. If significant alkane production occurs during stationary phase, inoculation to a high initial OD may increase alkane production. For this test, we inoculated XL-1 blue (to a final OD600 in a range of 0.01 to 10) in M9 production media (in covered glass culture tubes). We extracted from these cultures after 16 hours of growth. <br />
[[File:Washington_alkaneODcurve.png|center|500px|thumb|Greater Initial Cell Density Results in Higher Alkane Yield.]]<br />
Higher cell density cultures showed significantly higher alkane yields than lower cell density cultures. The increase of alkane yields at cell densities as high as 10 implies that high amounts of alkane production occurs during stationary phase. Therefore, alkane production scales with cell density. In addition, inducing alkane production during stationary phase decreases the amount of glucose going towards cellular growth instead of alkane production.<br />
=='''Final conditions'''==<br />
Using our optimized media & growth conditions(XL-1 blue inoculated to an OD600 of 10, in M9 media, in closed glass culture tubes) , we were able to improve total alkane yield 80 fold, from approximenly 2 mg/L (after 48 hrs) to approximently 170 mg/L(after 48 hrs).<br />
[[File:Washinton 2011 Optimization Quant.png|center|550px|thumb|Our optimized growth conditions resulted in an 80 fold increase in total alkane yield.]]<br />
=='''Production Curve'''==<br />
Ideally, alkane extraction and analysis would occur after all ( or essentially all) of the glucose in the media was used up. However, we didn't know how long it would take for our cells to use up all of the glucose. In order to determine the best time for extraction, we extracted alkanes from M9 production cultures that had been producing for 6, 24, 48, and 72 hours. Three cultures were used for each timepoint.<br />
[[file:Washington_Productioncurve.png|center|550px|thumb|Total alkane yield over time under optimized conditions.]]<br />
The 24 hour timepoint was essentially identical to the 48 and 72 hour timepoints. This implies that our cells are using up 30g/L glucose sometime before 24 hours. Future work will consist of increasing glucose concentration, and increasing inoculated cell density in order to maximize alkane production per culture per day.<br />
=='''Alternate Carbon Sources'''==<br />
While previous tests were done using glucose as the sole carbon source, it would be helpful to be able to use carbon sources that can be considered waste sources. There is a large amount of interest in using non-simple sugar carbon sources, such as cellulosic biomass, and even using carbon dioxide directly. However, these carbon sources cannot be easily utilized by ''E. coli''. Glycerol is a 3-carbon triol that is a waste product in the production of current generation biodiesel(fatty acid methyl esters(FAMEs). Glycerol can be used by ''E. coli'' as a sole carbon source, so we tried to use the PetroBrick to convert glycerol into diesel. We performed standard alkane production/analysis, with the second production media being either [https://2011.igem.org/Team:Washington/alkanebiosynthesis#Current_Protocol_for_100mL_of_Media standard M9 glucose media], or M9 media varients containing an equal mass of glycerol instead of glucose, or media without any carbon source.<br />
<br />
The glucose media resulted in higher total alkane yield than the glycerol media(240 mg/L vs. 56.3 mg/L), but this may be due to media conditions being optimized for glucose as a carbon source insteam od glycerol. Further optimization may be able to improve on alkane yield when using glycerol as a carbon source. In media without carbon, we observed negligable alkane production(4.7 mg/L). This indicates that alkane production is due to conversion of carbon in production media into alkanes, not due to the conversion of cellular biomass into alkanes. <br />
=='''References'''==<br />
Microbial Biosynthesis of Alkanes Andreas Schirmer, Mathew A. Rude, Xuezhi Li, Emanuela Popova and Stephen B. del Cardayre Science 30 July 2010: Vol. 329 no. 5991 pp. 559-562 http://www.sciencemag.org/content/329/5991/559</div>Hargemhttp://2011.igem.org/File:Washington_carbontest.pngFile:Washington carbontest.png2011-10-28T05:41:09Z<p>Hargem: uploaded a new version of &quot;File:Washington carbontest.png&quot;</p>
<hr />
<div></div>Hargemhttp://2011.igem.org/File:Washington_carbontest.pngFile:Washington carbontest.png2011-10-28T00:42:57Z<p>Hargem: </p>
<hr />
<div></div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/BackgroundTeam:Washington/Alkanes/Background2011-10-26T22:50:33Z<p>Hargem: /* The PetroBrick: A modular and open platform for the biological production of diesel fuel */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Background'''</big></big></big></big></center><br><br><br />
<br />
='''Petroleum, an Unfortunate Necessity'''=<br />
[[Image:Washington2011_PetroUsage.png|border|450px|right|thumb|Petroleum may be a necessity for modern civilization, but current extraction methods are unsustainable and non-cyclical in the long run.]]<br />
<br />
Modern society is completely dependent on petroleum based fuels. Automobiles are slowly transitioning towards electric power. However, for the foreseeable future, batteries will not be able to hold the energy needed for applications that require long range (e.g. jet planes, maritime shipping, and long range trucking) or high horsepower (e.g. agriculture, construction, industry). Without the use of petroleum, society as we know it would crumble. Petroleum is not a viable long term fuel because it a non-renewable resource. When petroleum based fuels are combusted, CO<sub>2</sub> is released into the atmosphere. Using current technology, it is impossible to turn this carbon dioxide back into fuel, meaning that the amount of petroleum based fuel is a finite commodity. In addition, this excess carbon dioxide is a potent greenhouse gas that contributes to global warming.<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
='''Todays biofuels are ''renewable'', but do not work as "drop-in" replacements'''=<br />
<br />
<br />
[[Image:Washington2011_BiofuelsAreRenewable.png|right|450px|frameless|thumb|Since carbon in biofuels can be derived from photosynthesis, biofuels are renewable. However, current biofuels have clear and problematic limits concerning energy production.]]<br />
<br />
Many different attempts have been made to produce a renewable, biologically derived fuel that would alleviate both the limited supply and emissions issues presented by petroleum based fuels. These efforts include alcohols (ethanol, butanol and other, higher alcohols), and biodiesel. Like petroleum based fuels, biofuels consist of combustible molecules that emit carbon dioxide. However, unlike petroleum based fuels, biofuels are renewable. CO<sub>2</sub> can be converted into more biofuel by feeding biofuel producing microbes (bacteria, yeast) photosynthetically derived plant biomass. Since the amount of CO<sub>2</sub> produced by burning a biofuel cannot exceed the CO<sub>2</sub> incorporated into plant biomass, a biofuel can be used indefinitely without any net carbon emissions.<br />
However, current biofuels consist of drastically different compounds from those found in petroleum. Petroleum consists of mostly long-chain length alkanes consisting of long hydrocarbon chains. Current biofuels contain either alcohols or long chain esters (biodiesel). Both of these molecules contain oxygen, which dramatically changes chemical properties. Both alcohols and biodiesel are more corrosive than unreactive alkanes. Alcohols are highly corrosive, both in pipelines ( [[#References | [1]]] ), and in engines not designed for the use of alcohols, even at concentrations as low as 20%( [[#References | [2]]] ). The corrosive property of alcohols in pipelines means that ethanol (the main alcohol in widespread use) is transported in vehicles (mostly by train), as opposed to by cheaper and less energy intensive pipelines( [[#References | [1]]] ). Transport of alcohols by pipeline would require retrofitting the entire fuel distribution infrastructure. The Fatty Acid Methyl Esters(FAMEs) in biodiesel are not directly as corrosive as alcohols, but can be biodegraded by anaerobic bacteria, producing hydrogen sulfide and other acids( [[#References | [3]]] ). Biodiesel has a higher freezing point than diesel, causing engine fuel filter clogging at low temperatures( [[#References | [4]]] ). Ethanol suffers from a much lower energy density than diesel(21.27-23.56 MJ/L vs 32.36-34.66 MJ/L)( [[#References | [5]]] ), resulting in lower gas mileage. The table below shows selected chemical properties of diesel, as well as the common biofuels ethanol, butanol, and biodiesel( data from [[#References | [5,6,9-12]]]). <br />
{| {{table}}<br />
| align="center" style="background:#f0f0f0;"|'''Property'''<br />
| align="center" style="background:#f0f0f0;"|'''Diesel'''<br />
| align="center" style="background:#f0f0f0;"|'''Ethanol'''<br />
| align="center" style="background:#f0f0f0;"|'''Biodiesel'''<br />
| align="center" style="background:#f0f0f0;"|'''n-Butanol'''<br />
|-<br />
| Specific gravity @ 15.5°C||0.85||0.794||0.88||.81(@20°C)<br />
|-<br />
| Density @ 15.5°C(g/L)||848.25||792.05||878.09||808.8(@20°C)<br />
|-<br />
| Energy Density(MJ/L)||32.36-34.66 ||21.27-23.56 ||33.32-35.66||27<br />
|-<br />
| Cetane number||40-55||0-54||48-65||17<br />
|-<br />
| Freezing point(°C)||-40 - -1||-114||-3 -19||-89.3<br />
|-<br />
| Viscosity( @ 20°C)(mm<sup>2</sup>/s)||2.8-5.0||1.5||6.4-6.6<br />
|-<br />
| Flash point(°C)||60-80||12.8||100-170||35<br />
|-<br />
| <br />
|} <br />
<br />
<br />
----<br />
<br />
='''The Ideal Fuel is Microbially-Produced Diesel'''=<br />
<br />
[[Image:Washington2011_PetroBiofuel.png|frameless|left|400px]]<br />
:::::::::::::::::<p>The ideal fuel would be compatible with modern engines and infrastructure, and also be able to be produced in a renewable manner. No current biofuel has properties identical enough to that of diesel to be able to fully integrate with current engines and infrastructure. No known alternative fuel is able to match the chemical properties of diesel. Currently, the only way to renewably produce a fuel with the chemical properties and compatibility of diesel would be to make a biofuel with a composition identical to that of diesel. This would require a biological pathway that is able to produce alkanes, the main class of compounds in diesel. Alkanes are simple chains of carbon and hydrogen. The majority of the alkanes found in diesel have a carbon chain of 10 to 20 carbons long. Alkanes make up approximately 62% of jet diesel (a fairly representative diesel fuel)([[#References | [7]]]). This 62% includes 34% straight chain alkanes that contain only one linear chain, and 28% branched chain alkanes that contain 1 or more carbon branches. The remaining 38% consists mostly of cyclic and aromatic hydrocarbons. If long (10+) chain length alkanes could be biologically produced, it would allow for the production of a fuel that is both renewable and fully compatible with current engines and infrastructure.</p><br />
<br />
<br />
----<br />
<br />
='''The Solution: a Microbial Alkane Production Pathway'''=<br />
[[Image:Washington2011_AlkaneAndBackCycle.png|right|400px|frameless|thumb| AAR converts the fatty acid intermediate Acyl-ACP into an aldehyde, which is decarbonylated into an alkane by ADC. Acyl-ACPs are naturally produced by all organisms, which increases host choice considerably.<br />
A recent study([[#References | [8]]]),has shown the production of long chain length alkanes in ''E. coli'' using two genes found in many cyanobacteria species. The first gene codes for [http://partsregistry.org/Part:BBa_K590032 Acyl-ACP Reductase] (AAR) which reduces long chain length acyl-ACPs into the corresponding fatty aldehydes. Acyl-ACPs are essential intermediates in fatty acid biosynthesis in every known organism, meaning that this system can work in a wide range of organisms. This long chain fatty acid acts as a substrate for [http://partsregistry.org/Part:BBa_K590031 Aldehyde Decarbonylase] (ADC), the enzyme that removes the carbonyl group (C=O) from the fatty aldehyde, yielding an alkane one carbon shorter than the original Acyl-ACP and a molecule of formate. Since the vast majority of the fatty acyl-ACPs produced by ''E. coli'' have an even chain length, this system produces detectable amounts of only odd chain length alkanes. This study reported production of the C13, C15, and C17 alkanes, as well as the C17 alkene (unsaturated hydrocarbon). This chain length range falls well within the range of those found in diesel, so this system is theoretically able to make the alkane portion of a fuel compatible with current engines and infrastructure.<br />
<br />
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<br />
='''The PetroBrick: A modular and open platform for the biological production of diesel fuel'''=<br />
<br />
[[File:Washington PetroBrick.png|200px|frameless|border|left|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
<br />
<br />
<br />
The goal of our "'''''Make It: Diesel Production'''''" portion of this summer's iGEM project is to convert this recently discovered set of enzymes ([http://partsregistry.org/Part:BBa_K590032 AAR] and [http://partsregistry.org/Part:BBa_K590031 ADC]) for microbial alkane production into an open and modular platform for iGEM teams to develop into a robust replacement for petrochemical fuels. Our alkane production system is specifically designed to be easily improved upon, and we have started work on improving this open system, both by increasing alkane yields and by changing the product produced. In addition, we have started to move this system into an alternative chassis, yeast.<br />
<br />
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<br />
==References==<br />
1. http://ourenergypolicy.org/docs/2/biofuels-taskforce.pdf<br />
<br />
2. http://www.environment.gov.au/atmosphere/fuelquality/publications/2000hours-vehicle-fleet/pubs/2000-hours-vehicles.pdf<br />
<br />
3. Anaerobic Metabolism of Biodiesel and Its Impact on Metal Corrosion<br />
Deniz F. Aktas, Jason S. Lee, Brenda J. Little, Richard I. Ray, Irene A. Davidova, Christopher N. Lyles, Joseph M. Suflita Energy & Fuels 2010 24 (5), 2924-2928(http://pubs.acs.org/doi/full/10.1021/ef100084j)<br />
<br />
4. http://www.mda.state.mn.us/news/publications/renewable/biodiesel/biodieselcoldissues.pdf<br />
<br />
5. http://www.afdc.energy.gov/afdc/pdfs/fueltable.pdf<br />
<br />
6. The viscosities of three biodiesel fuels at temperatures up to 300°C<br />
R.E. Tate, K.C. Watts, C.A.W. Allen K.I. Wilkie Fuel 2006 85, 1010-1015 (https://netfiles.uiuc.edu/mccrady/shared/Biodiesel/The%20viscosities%20of%20three%20biodiesel%20fuels%20at%20temperatures%20up%20to%20300%208C.pdf)<br />
<br />
7. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
8. Microbial Biosynthesis of Alkanes Andreas Schirmer, Mathew A. Rude, Xuezhi Li, Emanuela Popova and Stephen B. del Cardayre Science 30 July 2010: Vol. 329 no. 5991 pp. 559-562 http://www.sciencemag.org/content/329/5991/559<br />
<br />
9.http://msdssearch.dow.com/PublishedLiteratureDOWCOM/dh_0119/0901b803801195a5.pdf?filepath=oxysolvents/pdfs/noreg/327-00014.pdf&fromPage=GetDoc<br />
<br />
10.Metabolic engineering of Escherichia coli for 1-butanol production Shota Atsumi, Anthony F. Cann, Michael R. Connor, Claire R. Shen, Kevin M. Smith, Mark P. Brynildsen, Katherine J.Y. Chou, Taizo Hanai1, James C. Liao Metabolic Engineering Volume 10, Issue 6, November 2008, Pages 305-311<br />
<br />
11. http://www.nrel.gov/vehiclesandfuels/pdfs/sr368051.pdf<br />
<br />
12. http://www.arpltd.com/n-butano.htm</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/BackgroundTeam:Washington/Alkanes/Background2011-10-26T22:50:07Z<p>Hargem: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Background'''</big></big></big></big></center><br><br><br />
<br />
='''Petroleum, an Unfortunate Necessity'''=<br />
[[Image:Washington2011_PetroUsage.png|border|450px|right|thumb|Petroleum may be a necessity for modern civilization, but current extraction methods are unsustainable and non-cyclical in the long run.]]<br />
<br />
Modern society is completely dependent on petroleum based fuels. Automobiles are slowly transitioning towards electric power. However, for the foreseeable future, batteries will not be able to hold the energy needed for applications that require long range (e.g. jet planes, maritime shipping, and long range trucking) or high horsepower (e.g. agriculture, construction, industry). Without the use of petroleum, society as we know it would crumble. Petroleum is not a viable long term fuel because it a non-renewable resource. When petroleum based fuels are combusted, CO<sub>2</sub> is released into the atmosphere. Using current technology, it is impossible to turn this carbon dioxide back into fuel, meaning that the amount of petroleum based fuel is a finite commodity. In addition, this excess carbon dioxide is a potent greenhouse gas that contributes to global warming.<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
='''Todays biofuels are ''renewable'', but do not work as "drop-in" replacements'''=<br />
<br />
<br />
[[Image:Washington2011_BiofuelsAreRenewable.png|right|450px|frameless|thumb|Since carbon in biofuels can be derived from photosynthesis, biofuels are renewable. However, current biofuels have clear and problematic limits concerning energy production.]]<br />
<br />
Many different attempts have been made to produce a renewable, biologically derived fuel that would alleviate both the limited supply and emissions issues presented by petroleum based fuels. These efforts include alcohols (ethanol, butanol and other, higher alcohols), and biodiesel. Like petroleum based fuels, biofuels consist of combustible molecules that emit carbon dioxide. However, unlike petroleum based fuels, biofuels are renewable. CO<sub>2</sub> can be converted into more biofuel by feeding biofuel producing microbes (bacteria, yeast) photosynthetically derived plant biomass. Since the amount of CO<sub>2</sub> produced by burning a biofuel cannot exceed the CO<sub>2</sub> incorporated into plant biomass, a biofuel can be used indefinitely without any net carbon emissions.<br />
However, current biofuels consist of drastically different compounds from those found in petroleum. Petroleum consists of mostly long-chain length alkanes consisting of long hydrocarbon chains. Current biofuels contain either alcohols or long chain esters (biodiesel). Both of these molecules contain oxygen, which dramatically changes chemical properties. Both alcohols and biodiesel are more corrosive than unreactive alkanes. Alcohols are highly corrosive, both in pipelines ( [[#References | [1]]] ), and in engines not designed for the use of alcohols, even at concentrations as low as 20%( [[#References | [2]]] ). The corrosive property of alcohols in pipelines means that ethanol (the main alcohol in widespread use) is transported in vehicles (mostly by train), as opposed to by cheaper and less energy intensive pipelines( [[#References | [1]]] ). Transport of alcohols by pipeline would require retrofitting the entire fuel distribution infrastructure. The Fatty Acid Methyl Esters(FAMEs) in biodiesel are not directly as corrosive as alcohols, but can be biodegraded by anaerobic bacteria, producing hydrogen sulfide and other acids( [[#References | [3]]] ). Biodiesel has a higher freezing point than diesel, causing engine fuel filter clogging at low temperatures( [[#References | [4]]] ). Ethanol suffers from a much lower energy density than diesel(21.27-23.56 MJ/L vs 32.36-34.66 MJ/L)( [[#References | [5]]] ), resulting in lower gas mileage. The table below shows selected chemical properties of diesel, as well as the common biofuels ethanol, butanol, and biodiesel( data from [[#References | [5,6,9-12]]]). <br />
{| {{table}}<br />
| align="center" style="background:#f0f0f0;"|'''Property'''<br />
| align="center" style="background:#f0f0f0;"|'''Diesel'''<br />
| align="center" style="background:#f0f0f0;"|'''Ethanol'''<br />
| align="center" style="background:#f0f0f0;"|'''Biodiesel'''<br />
| align="center" style="background:#f0f0f0;"|'''n-Butanol'''<br />
|-<br />
| Specific gravity @ 15.5°C||0.85||0.794||0.88||.81(@20°C)<br />
|-<br />
| Density @ 15.5°C(g/L)||848.25||792.05||878.09||808.8(@20°C)<br />
|-<br />
| Energy Density(MJ/L)||32.36-34.66 ||21.27-23.56 ||33.32-35.66||27<br />
|-<br />
| Cetane number||40-55||0-54||48-65||17<br />
|-<br />
| Freezing point(°C)||-40 - -1||-114||-3 -19||-89.3<br />
|-<br />
| Viscosity( @ 20°C)(mm<sup>2</sup>/s)||2.8-5.0||1.5||6.4-6.6<br />
|-<br />
| Flash point(°C)||60-80||12.8||100-170||35<br />
|-<br />
| <br />
|} <br />
<br />
<br />
----<br />
<br />
='''The Ideal Fuel is Microbially-Produced Diesel'''=<br />
<br />
[[Image:Washington2011_PetroBiofuel.png|frameless|left|400px]]<br />
:::::::::::::::::<p>The ideal fuel would be compatible with modern engines and infrastructure, and also be able to be produced in a renewable manner. No current biofuel has properties identical enough to that of diesel to be able to fully integrate with current engines and infrastructure. No known alternative fuel is able to match the chemical properties of diesel. Currently, the only way to renewably produce a fuel with the chemical properties and compatibility of diesel would be to make a biofuel with a composition identical to that of diesel. This would require a biological pathway that is able to produce alkanes, the main class of compounds in diesel. Alkanes are simple chains of carbon and hydrogen. The majority of the alkanes found in diesel have a carbon chain of 10 to 20 carbons long. Alkanes make up approximately 62% of jet diesel (a fairly representative diesel fuel)([[#References | [7]]]). This 62% includes 34% straight chain alkanes that contain only one linear chain, and 28% branched chain alkanes that contain 1 or more carbon branches. The remaining 38% consists mostly of cyclic and aromatic hydrocarbons. If long (10+) chain length alkanes could be biologically produced, it would allow for the production of a fuel that is both renewable and fully compatible with current engines and infrastructure.</p><br />
<br />
<br />
----<br />
<br />
='''The Solution: a Microbial Alkane Production Pathway'''=<br />
[[Image:Washington2011_AlkaneAndBackCycle.png|right|400px|frameless|thumb| AAR converts the fatty acid intermediate Acyl-ACP into an aldehyde, which is decarbonylated into an alkane by ADC. Acyl-ACPs are naturally produced by all organisms, which increases host choice considerably.<br />
A recent study([[#References | [8]]]),has shown the production of long chain length alkanes in ''E. coli'' using two genes found in many cyanobacteria species. The first gene codes for [http://partsregistry.org/Part:BBa_K590032 Acyl-ACP Reductase] (AAR) which reduces long chain length acyl-ACPs into the corresponding fatty aldehydes. Acyl-ACPs are essential intermediates in fatty acid biosynthesis in every known organism, meaning that this system can work in a wide range of organisms. This long chain fatty acid acts as a substrate for [http://partsregistry.org/Part:BBa_K590031 Aldehyde Decarbonylase] (ADC), the enzyme that removes the carbonyl group (C=O) from the fatty aldehyde, yielding an alkane one carbon shorter than the original Acyl-ACP and a molecule of formate. Since the vast majority of the fatty acyl-ACPs produced by ''E. coli'' have an even chain length, this system produces detectable amounts of only odd chain length alkanes. This study reported production of the C13, C15, and C17 alkanes, as well as the C17 alkene (unsaturated hydrocarbon). This chain length range falls well within the range of those found in diesel, so this system is theoretically able to make the alkane portion of a fuel compatible with current engines and infrastructure.<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
='''The PetroBrick: A modular and open platform for the biological production of diesel fuel'''=<br />
<br />
[[File:Washington PetroBrick.png|200px|frameless|border|left|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
<br />
<br />
<br />
The goal of our "'''''Make It: Diesel Production'''''" portion of this summer's iGEM project is to turn convert this recently discovered set of enzymes ([http://partsregistry.org/Part:BBa_K590032 AAR] and [http://partsregistry.org/Part:BBa_K590031 ADC]) for microbial alkane production into an open and modular platform for iGEM teams to develop into a robust replacement for petrochemical fuels. Our alkane production system is specifically designed to be easily improved upon, and we have started work on improving this open system, both by increasing alkane yields and by changing the product produced. In addition, we have started to move this system into an alternative chassis, yeast.<br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
<br />
----<br />
<br />
<br />
==References==<br />
1. http://ourenergypolicy.org/docs/2/biofuels-taskforce.pdf<br />
<br />
2. http://www.environment.gov.au/atmosphere/fuelquality/publications/2000hours-vehicle-fleet/pubs/2000-hours-vehicles.pdf<br />
<br />
3. Anaerobic Metabolism of Biodiesel and Its Impact on Metal Corrosion<br />
Deniz F. Aktas, Jason S. Lee, Brenda J. Little, Richard I. Ray, Irene A. Davidova, Christopher N. Lyles, Joseph M. Suflita Energy & Fuels 2010 24 (5), 2924-2928(http://pubs.acs.org/doi/full/10.1021/ef100084j)<br />
<br />
4. http://www.mda.state.mn.us/news/publications/renewable/biodiesel/biodieselcoldissues.pdf<br />
<br />
5. http://www.afdc.energy.gov/afdc/pdfs/fueltable.pdf<br />
<br />
6. The viscosities of three biodiesel fuels at temperatures up to 300°C<br />
R.E. Tate, K.C. Watts, C.A.W. Allen K.I. Wilkie Fuel 2006 85, 1010-1015 (https://netfiles.uiuc.edu/mccrady/shared/Biodiesel/The%20viscosities%20of%20three%20biodiesel%20fuels%20at%20temperatures%20up%20to%20300%208C.pdf)<br />
<br />
7. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
8. Microbial Biosynthesis of Alkanes Andreas Schirmer, Mathew A. Rude, Xuezhi Li, Emanuela Popova and Stephen B. del Cardayre Science 30 July 2010: Vol. 329 no. 5991 pp. 559-562 http://www.sciencemag.org/content/329/5991/559<br />
<br />
9.http://msdssearch.dow.com/PublishedLiteratureDOWCOM/dh_0119/0901b803801195a5.pdf?filepath=oxysolvents/pdfs/noreg/327-00014.pdf&fromPage=GetDoc<br />
<br />
10.Metabolic engineering of Escherichia coli for 1-butanol production Shota Atsumi, Anthony F. Cann, Michael R. Connor, Claire R. Shen, Kevin M. Smith, Mark P. Brynildsen, Katherine J.Y. Chou, Taizo Hanai1, James C. Liao Metabolic Engineering Volume 10, Issue 6, November 2008, Pages 305-311<br />
<br />
11. http://www.nrel.gov/vehiclesandfuels/pdfs/sr368051.pdf<br />
<br />
12. http://www.arpltd.com/n-butano.htm</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/BackgroundTeam:Washington/Alkanes/Background2011-10-26T22:44:18Z<p>Hargem: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Background'''</big></big></big></big></center><br><br><br />
<br />
='''Petroleum, an Unfortunate Necessity'''=<br />
[[Image:Washington2011_PetroUsage.png|border|450px|right|thumb|Petroleum may be a necessity for modern civilization, but current extraction methods are unsustainable and non-cyclical in the long run.]]<br />
<br />
Modern society is completely dependent on petroleum based fuels. Automobiles are slowly transitioning towards electric power. However, for the foreseeable future, batteries will not be able to hold the energy needed for applications that require long range (e.g. jet planes, maritime shipping, and long range trucking) or high horsepower (e.g. agriculture, construction, industry). Without the use of petroleum, society as we know it would crumble. Petroleum is not a viable long term fuel because it a non-renewable resource. When petroleum based fuels are combusted, CO<sub>2</sub> is released into the atmosphere. Using current technology, it is impossible to turn this carbon dioxide back into fuel, meaning that the amount of petroleum based fuel is a finite commodity. In addition, this excess carbon dioxide is a potent greenhouse gas that contributes to global warming.<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
='''Todays biofuels are ''renewable'', but do not work as "drop-in" replacements'''=<br />
<br />
<br />
[[Image:Washington2011_BiofuelsAreRenewable.png|right|450px|frameless|thumb|Since carbon in biofuels can be derived from photosynthesis, biofuels are renewable. However, current biofuels have clear and problematic limits concerning energy production.]]<br />
<br />
Many different attempts have been made to produce a renewable, biologically derived fuel that would alleviate both the limited supply and emissions issues presented by petroleum based fuels. These efforts include alcohols (ethanol, butanol and other, higher alcohols), and biodiesel . Like petroleum based fuels, biofuels consist of combustible molecules that emit carbon dioxide. However, unlike petroleum based fuels, biofuels are renewable. CO<sub>2</sub> can be converted into more biofuel by feeding biofuel producing microbes (bacteria, yeast) photosynthetically derived plant biomass. Since the amount of CO<sub>2</sub> produced by burning a biofuel cannot exceed the CO<sub>2</sub> incorporated into plant biomass, a biofuel can be used indefinitely without any net carbon emissions.<br />
However, current biofuels consist of drastically different compounds from those found in petroleum. Petroleum consists of mostly long-chain length alkanes consisting of long hydrocarbon chains. Current biofuels contain either alcohols or long chain esters (biodiesel). Both of these molecules contain oxygen, which dramatically changes chemical properties. Both alcohols and biodiesel are more corrosive than unreactive alkanes. Alcohols are highly corrosive, both in pipelines ( [[#References | [1]]] ), and in engines not designed for the use of alcohols, even at concentrations as low as 20%( [[#References | [2]]] ). The corrosive property of alcohols in pipelines means that ethanol (the main alcohol in widespread use) is transported in vehicles (mostly by train), as opposed to by cheaper and less energy intensive pipelines( [[#References | [1]]] ). Transport of alcohols by pipeline would require retrofitting the entire fuel distribution infrastructure. The Fatty Acid Methyl Esters(FAMEs) in biodiesel are not directly as corrosive as alcohols, but can be biodegraded by anaerobic bacteria, producing hydrogen sulfide and other acids( [[#References | [3]]] ). Biodiesel has a higher freezing point than diesel, causing engine fuel filer clogging at low temperatures( [[#References | [4]]] ). Ethanol suffers from a much lower energy density than diesel(21.27-23.56 MJ/L vs 32.36-34.66 MJ/L)( [[#References | [5]]] ), resulting in lower gas mileage. The table below shows selected chemical properties of diesel, as well as the common biofuels ethanol, butanol, and biodiesel( data from [[#References | [5,6,9-12]]]). <br />
{| {{table}}<br />
| align="center" style="background:#f0f0f0;"|'''Property'''<br />
| align="center" style="background:#f0f0f0;"|'''Diesel'''<br />
| align="center" style="background:#f0f0f0;"|'''Ethanol'''<br />
| align="center" style="background:#f0f0f0;"|'''Biodiesel'''<br />
| align="center" style="background:#f0f0f0;"|'''n-Butanol'''<br />
|-<br />
| Specific gravity @ 15.5°C||0.85||0.794||0.88||.81(@20°C)<br />
|-<br />
| Density @ 15.5°C(g/L)||848.25||792.05||878.09||808.8(@20°C)<br />
|-<br />
| Energy Density(MJ/L)||32.36-34.66 ||21.27-23.56 ||33.32-35.66||27<br />
|-<br />
| Cetane number||40-55||0-54||48-65||17<br />
|-<br />
| Freezing point(°C)||-40 - -1||-114||-3 -19||-89.3<br />
|-<br />
| Viscosity( @ 20°C)(mm<sup>2</sup>/s)||2.8-5.0||1.5||6.4-6.6<br />
|-<br />
| Flash point(°C)||60-80||12.8||100-170||35<br />
|-<br />
| <br />
|} <br />
<br />
<br />
----<br />
<br />
='''The Ideal Fuel is Microbially-Produced Diesel'''=<br />
<br />
[[Image:Washington2011_PetroBiofuel.png|frameless|left|400px]]<br />
:::::::::::::::::<p>The ideal fuel would be compatible with modern engines and infrastructure, and also be able to be produced in a renewable manner. No current biofuel has the same identical enough to that of diesel to be able to fully integrate with current engines and infrastructure. No known alternative fuel is able to match the chemical properties of diesel. Currently, the only way to renewably produce a fuel with the chemical properties and compatibility of diesel would be to make a biofuel with a composition identical to that of diesel. This would require a biological pathway that is able to produce alkanes, the main class of compounds in diesel. Alkanes are simple chains of carbon and hydrogen. The majority of the alkanes found in diesel have a carbon chain of 10 to 20 carbons long. Alkanes make up approximately 62% of jet diesel (a fairly representative diesel fuel)([[#References | [7]]]). This 62% includes 34% straight chain alkanes that contain only one linear chain, and 28% branched chain alkanes that contain 1 or more carbon branches. The remaining 38% consists mostly of cyclic and aromatic hydrocarbons. If long (10+) chain length alkanes could be biologically produced, it would allow for the production of a fuel that is both renewable and fully compatible with current engines and infrastructure.</p><br />
<br />
<br />
----<br />
<br />
='''The Solution: a Microbial Alkane Production Pathway'''=<br />
[[Image:Washington2011_AlkaneAndBackCycle.png|right|400px|frameless|thumb|The use of AAR and ADC converts the fatty acid intermediate Acl-ACP into an aldehyde, then an alkane--biofuel. Acyl-ACP is naturally produced by all organisms, which increases host choice considerably]]<br />
A recent study([[#References | [8]]]),has shown the production of long chain length alkanes in ''E. coli'' using two genes found in many cyanobacteria species. The first gene codes for [http://partsregistry.org/Part:BBa_K590032 Acyl-ACP Reductase] (AAR) which reduces long chain length acyl-ACPs into the corresponding fatty aldehydes. Acyl-ACPs are essential intermediates in fatty acid biosynthesis in every known organism, meaning that this system can work in a wide range of organisms. This long chain fatty acid acts as a substrate for [http://partsregistry.org/Part:BBa_K590031 Aldehyde Decarbonylase] (ADC), the enzyme that removes the carbonyl group (C=O) from the fatty aldehyde, yielding an alkane one carbon shorter than the original Acyl-ACP and a molecule of formate. Since the vast majority of the fatty acyl-ACPs produced by ''E. coli'' have an even chain length, this system produces detectable amounts of only odd chain length alkanes. This study reported production of the C13, C15, and C17 alkanes, as well as the C17 alkene (unsaturated hydrocarbon). This chain length range falls well within the range of those found in diesel, so this system is theoretically able to make the alkane portion of a fuel compatible with current engines and infrastructure.<br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
='''The PetroBrick: A modular and open platform for the biological production of diesel fuel'''=<br />
<br />
[[File:Washington PetroBrick.png|200px|frameless|border|left|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
<br />
<br />
<br />
The goal of our "'''''Make It: Diesel Production'''''" portion of this summer's iGEM project is to turn convert this recently discovered set of enzymes ([http://partsregistry.org/Part:BBa_K590032 AAR] and [http://partsregistry.org/Part:BBa_K590031 ADC]) for microbial alkane production into an open and modular platform for iGEM teams to develop into a robust replacement for petrochemical fuels. Our alkane production system is specifically designed to be easily improved upon, and we have started work on improving this open system, both by increasing alkane yields and by changing the product produced. In addition, we have started to move this system into an alternative chassis, yeast.<br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
<br />
----<br />
<br />
<br />
==References==<br />
1. http://ourenergypolicy.org/docs/2/biofuels-taskforce.pdf<br />
<br />
2. http://www.environment.gov.au/atmosphere/fuelquality/publications/2000hours-vehicle-fleet/pubs/2000-hours-vehicles.pdf<br />
<br />
3. Anaerobic Metabolism of Biodiesel and Its Impact on Metal Corrosion<br />
Deniz F. Aktas, Jason S. Lee, Brenda J. Little, Richard I. Ray, Irene A. Davidova, Christopher N. Lyles, Joseph M. Suflita Energy & Fuels 2010 24 (5), 2924-2928(http://pubs.acs.org/doi/full/10.1021/ef100084j)<br />
<br />
4. http://www.mda.state.mn.us/news/publications/renewable/biodiesel/biodieselcoldissues.pdf<br />
<br />
5. http://www.afdc.energy.gov/afdc/pdfs/fueltable.pdf<br />
<br />
6. The viscosities of three biodiesel fuels at temperatures up to 300°C<br />
R.E. Tate, K.C. Watts, C.A.W. Allen K.I. Wilkie Fuel 2006 85, 1010-1015 (https://netfiles.uiuc.edu/mccrady/shared/Biodiesel/The%20viscosities%20of%20three%20biodiesel%20fuels%20at%20temperatures%20up%20to%20300%208C.pdf)<br />
<br />
7. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
8. Microbial Biosynthesis of Alkanes Andreas Schirmer, Mathew A. Rude, Xuezhi Li, Emanuela Popova and Stephen B. del Cardayre Science 30 July 2010: Vol. 329 no. 5991 pp. 559-562 http://www.sciencemag.org/content/329/5991/559<br />
<br />
9.http://msdssearch.dow.com/PublishedLiteratureDOWCOM/dh_0119/0901b803801195a5.pdf?filepath=oxysolvents/pdfs/noreg/327-00014.pdf&fromPage=GetDoc<br />
<br />
10.Metabolic engineering of Escherichia coli for 1-butanol production Shota Atsumi, Anthony F. Cann, Michael R. Connor, Claire R. Shen, Kevin M. Smith, Mark P. Brynildsen, Katherine J.Y. Chou, Taizo Hanai1, James C. Liao Metabolic Engineering Volume 10, Issue 6, November 2008, Pages 305-311<br />
<br />
11. http://www.nrel.gov/vehiclesandfuels/pdfs/sr368051.pdf<br />
<br />
12. http://www.arpltd.com/n-butano.htm</div>Hargemhttp://2011.igem.org/Team:WashingtonTeam:Washington2011-10-26T22:43:35Z<p>Hargem: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<html><meta name="google-site-verification" content="fg3_xZB6BF10NZTT7oSIbF6AmRx0o-b-VZdgok0O3Ok" /></html><br />
=<center>'''Make It or Break It: <br/> Diesel Production and Gluten Destruction, the Synthetic Biology Way'''</center>=<br />
<br />
<center>Synthetic biology holds great promise regarding the production of important compounds, and the degradation of harmful ones. This summer, we harnessed the power of synthetic biology to meet the world’s needs for fuel and medicine.</center><br />
<br/><br />
[[Image:Washington_Fire.jpg|left|320px|borderless|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[Image:Washington_Bottle.jpg|right|200px|borderless|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
<br />
[https://2011.igem.org/Team:Washington/Alkanes/Background '''Make It: Diesel Production'''] We constructed a strain of ''Escherichia coli'' that produces a variety of alkanes, the main constituents of diesel fuel, by introducing a pair of genes recently shown to convert fatty acid synthesis intermediates into alkanes. <br />
<br />
[https://2011.igem.org/Team:Washington/Celiacs/Background '''Break It: Gluten Destruction'''] We identified a protease with gluten-degradation potential, and then reengineered it to have greatly increased gluten-degrading activity, allowing for the breakdown of gluten in the digestive track when taken in pill form. <br />
<br />
[https://2011.igem.org/Team:Washington/Magnetosomes/Background '''iGEM Toolkits'''] To enable next-generation cloning of standard biological parts, we built BioBrick vectors optimized for Gibson assembly and used them to create the Magnetosome Toolkit: a set of 18 genes from an essential operon in magnetotactic bacteria which we are characterizing to create magnetic ''E. coli''.<br />
<br />
<br />
[[File:Washington_Spacer.jpg|1px]]<br />
[[Image:UW Diesel Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Toolkits Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Gluten Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
[[File:Washington_Spacer.jpg|5px]]<br />
<br />
<br />
<br/><br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_OSLI.png|frameless|border|link=http://www.osli.ca|Oil Sands Leadership Intiative]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_UniversitySeal.gif|frameless|border|110px|link=http://www.washington.edu|University of Washington]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_Anaspec.gif|frameless|border|120px|link=http://www.anaspec.com|Anaspec]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_ARPA-E_Logo.png|frameless|border|link=http://arpa-e.energy.gov/ProgramsProjects/Electrofuels.aspx|Advanced Research Projects Agency - Energy]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington2011_Hhmi_362_72.jpg|link=http://www.hhmi.org/|Howard Hughes Medical Institute]]</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/FabH2Team:Washington/Alkanes/Future/FabH22011-10-26T22:41:18Z<p>Hargem: /* FabBrick Induction Results in Even Chain Length Alkane Production */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
__NOTOC__<br />
<br />
<big><big><big>'''<center>Alternative Alkane Products</center>'''</big></big></big><br />
<br />
=Background=<br />
[[File:Washington_branched_CoA.png|right|450px|thumb|The branched CoA substrates used by FabH2]]<br />
The basic alkane production system is incapable of making branched chain alkanes, as ''E. coli'' normally only makes straight chain fatty acids. In addition, our basic system is only able to make odd chain length alkanes, as E. coli only normally make even chain length fatty acids. We thought that if we could introduce an enzyme into PetroBrick expressing E. coli that results in the production of branched chain or odd chain length Fatty acids, we could extend the products produced by the PetroBrick to include branched chain or even change length alkanes. One protein whose expression has been previously shown ([[#References | [1]]]) to cause ''E. coli'' to produce branched chain and odd chain length fatty acids is FabH2, a FabH homolog from ''Bacillus subtilis''. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in even chain length linear fatty acids. However, FabH2 has been hypothesized to initiate fatty acid biosynthesis using Isobutyryl-CoA and Isovaleryl-CoA( products from Valine and Leucine degredation), resulting in 2-methyl branched fatty acid production, as well as using a 3-carbon propanoyl-CoA, resulting in odd chain length fatty acids. If we could use FabH2 and the Petrobrick to get ''E. coli'' to produce both alkanes and branched chain or odd chain length fatty acids, we should theoretically be able to produce 2-methyl branched alkanes or even chain length fatty acids.<br />
<br />
=Methods=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2 ] was constructed from oligos( refer to [https://2011.igem.org/Team:Washington/Protocols/gene_assembly protocol]). FabH2 was then amplified to add an [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 Elowitz standard RBS]. This RBS-FabH2 construct was cloned into the 3' end of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] to express [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2 as well as AAR and ADC]. In addition, we cloned fabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTG inducible vector].<br />
<br />
=FabH2 is Toxic to ''E. coli''=<br />
Throughout our experiments, we observed that cells expressing FabH2 grew significantly slower than any of our other alkane producing cells. In order to quantify this effect, we measured OD600 for alkane production constructs every 24 hours for a 72 hour period, producing a growth curve.<br />
[[File:Washington growthcurve.png|center|450px|thumb|Growth curve showing that cells overexpressing FabH2 are growth deficient. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 ADC] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 AAR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 DrR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2]<br />
]]<br />
After 24 hours, FabH2 producing cells had barely grown at all, indicating a severe growth deficiency. The cells were able to rapidly grow after 24 hours, presumably due to a mutation that counteracted the negative effects of FabH2. This toxicity could be due to branched chain fatty acid production, or due to the activity of FabH2 on straight chain substrates affecting cellular metabolism. This severe growth deficiency implies that FabH2 is being expressed, and has activity that is detrimental to cell growth. Due to FabH2's toxicity, any time that we co-expressed FabH2 and the PetroBrick on a high copy number, strong promter expression vector, total alkane yield was decimated to under 10 mg/L. Reduction of this toxicity should allow us to determine the effects of FabH2 production on alkane biosynthesis.<br />
<br />
=Reducing the Toxicity of FabH2.=<br />
We hypothesized that if we could reduce the toxicity of FabH2, we could see the production of alternative alkane products. The original FabH2 system placed the FabH2 gene under the control of a strong constitutive promoter. We thought that if we could reduce FabH2 expression, we could decrease toxicity and be able to see the effects of FabH2 production on the alkanes produced by the PetroBrick. We cloned FabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number 3k3 IPTG inducible vector]. By reducing copy number approximately 10 fold, and by having the ability to control expression, we thought that we could lessen toxicity and observe even chain length alkane production. We called this inducible, low expression construct the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590064 FabBrick], a modular add-on to the standard PetroBrick.<br />
<br />
=FabBrick Induction Results in Even Chain Length Alkane Production=<br />
The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/PetroBrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When the FabBrick is induced, we see a new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that of alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinantly, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels.<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
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[[Image:WashingtonC16zoomed.png|middle|400px|thumb|Zoomed in spectrum. Note the parent ion at a mass of 226.]]<br />
<br />
<br />
<br />
<br />
==References==<br />
1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.<br />
<br />
2. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
3. Branched-chain fatty acid biosynthesis in Escherichia coli. Smirnova N, Reynolds KA. J Ind Microbiol Biotechnol. 2001 Oct;27(4):246-51.<br />
<br />
=Parts Submitted=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2] This part contains the FabH2 coding sequence, codon optimized for ''E. coli''<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2-ADC-AAR] This part expresses FabH2, as well as AAR and ADC, the two genes responsible for alkane production. Note that this part didn't work as expected, likely due to FabH2 toxicity. <br />
<br />
[http://partsregistry.org/Part:BBa_K590064 The FabBrick] This part expresses FabH2 in a low copynumber, inducible vector. By co-expressing this part with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick], we were able to get even chain length alkane production.</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/FabH2Team:Washington/Alkanes/Future/FabH22011-10-26T22:38:36Z<p>Hargem: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
__NOTOC__<br />
<br />
<big><big><big>'''<center>Alternative Alkane Products</center>'''</big></big></big><br />
<br />
=Background=<br />
[[File:Washington_branched_CoA.png|right|450px|thumb|The branched CoA substrates used by FabH2]]<br />
The basic alkane production system is incapable of making branched chain alkanes, as ''E. coli'' normally only makes straight chain fatty acids. In addition, our basic system is only able to make odd chain length alkanes, as E. coli only normally make even chain length fatty acids. We thought that if we could introduce an enzyme into PetroBrick expressing E. coli that results in the production of branched chain or odd chain length Fatty acids, we could extend the products produced by the PetroBrick to include branched chain or even change length alkanes. One protein whose expression has been previously shown ([[#References | [1]]]) to cause ''E. coli'' to produce branched chain and odd chain length fatty acids is FabH2, a FabH homolog from ''Bacillus subtilis''. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in even chain length linear fatty acids. However, FabH2 has been hypothesized to initiate fatty acid biosynthesis using Isobutyryl-CoA and Isovaleryl-CoA( products from Valine and Leucine degredation), resulting in 2-methyl branched fatty acid production, as well as using a 3-carbon propanoyl-CoA, resulting in odd chain length fatty acids. If we could use FabH2 and the Petrobrick to get ''E. coli'' to produce both alkanes and branched chain or odd chain length fatty acids, we should theoretically be able to produce 2-methyl branched alkanes or even chain length fatty acids.<br />
<br />
=Methods=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2 ] was constructed from oligos( refer to [https://2011.igem.org/Team:Washington/Protocols/gene_assembly protocol]). FabH2 was then amplified to add an [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 Elowitz standard RBS]. This RBS-FabH2 construct was cloned into the 3' end of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] to express [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2 as well as AAR and ADC]. In addition, we cloned fabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTG inducible vector].<br />
<br />
=FabH2 is Toxic to ''E. coli''=<br />
Throughout our experiments, we observed that cells expressing FabH2 grew significantly slower than any of our other alkane producing cells. In order to quantify this effect, we measured OD600 for alkane production constructs every 24 hours for a 72 hour period, producing a growth curve.<br />
[[File:Washington growthcurve.png|center|450px|thumb|Growth curve showing that cells overexpressing FabH2 are growth deficient. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 ADC] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 AAR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 DrR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2]<br />
]]<br />
After 24 hours, FabH2 producing cells had barely grown at all, indicating a severe growth deficiency. The cells were able to rapidly grow after 24 hours, presumably due to a mutation that counteracted the negative effects of FabH2. This toxicity could be due to branched chain fatty acid production, or due to the activity of FabH2 on straight chain substrates affecting cellular metabolism. This severe growth deficiency implies that FabH2 is being expressed, and has activity that is detrimental to cell growth. Due to FabH2's toxicity, any time that we co-expressed FabH2 and the PetroBrick on a high copy number, strong promter expression vector, total alkane yield was decimated to under 10 mg/L. Reduction of this toxicity should allow us to determine the effects of FabH2 production on alkane biosynthesis.<br />
<br />
=Reducing the Toxicity of FabH2.=<br />
We hypothesized that if we could reduce the toxicity of FabH2, we could see the production of alternative alkane products. The original FabH2 system placed the FabH2 gene under the control of a strong constitutive promoter. We thought that if we could reduce FabH2 expression, we could decrease toxicity and be able to see the effects of FabH2 production on the alkanes produced by the PetroBrick. We cloned FabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number 3k3 IPTG inducible vector]. By reducing copy number approximately 10 fold, and by having the ability to control expression, we thought that we could lessen toxicity and observe even chain length alkane production. We called this inducible, low expression construct the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590064 FabBrick], a modular add-on to the standard PetroBrick.<br />
<br />
=FabBrick Induction Results in Even Chain Length Alkane Production=<br />
The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/PetroBrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When FabH2 is induced, we see a new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that of alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinantly, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels.<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]<br />
<br />
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<br />
[[Image:WashingtonC16zoomed.png|middle|400px|thumb|Zoomed in spectrum. Note the parent ion at a mass of 226.]]<br />
<br />
<br />
<br />
<br />
==References==<br />
1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.<br />
<br />
2. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
3. Branched-chain fatty acid biosynthesis in Escherichia coli. Smirnova N, Reynolds KA. J Ind Microbiol Biotechnol. 2001 Oct;27(4):246-51.<br />
<br />
=Parts Submitted=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2] This part contains the FabH2 coding sequence, codon optimized for ''E. coli''<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2-ADC-AAR] This part expresses FabH2, as well as AAR and ADC, the two genes responsible for alkane production. Note that this part didn't work as expected, likely due to FabH2 toxicity. <br />
<br />
[http://partsregistry.org/Part:BBa_K590064 The FabBrick] This part expresses FabH2 in a low copynumber, inducible vector. By co-expressing this part with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick], we were able to get even chain length alkane production.</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/FabH2Team:Washington/Alkanes/Future/FabH22011-10-26T22:35:44Z<p>Hargem: /* FabBrick Induction Results in Even Chain Length Alkane Production */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
__NOTOC__<br />
<br />
<big><big><big>'''<center>Alternative Alkane Products</center>'''</big></big></big><br />
<br />
=Background=<br />
[[File:Washington_branched_CoA.png|right|450px|thumb|The branched CoA substrates used by FabH2]]<br />
The basic alkane production system is incapable of making branched chain alkanes, as ''E. coli'' normally only makes straight chain fatty acids. In addition, our basic system is only able to make odd chain length alkanes, as E. coli only normally make even chain length fatty acids. We thought that if we could introduce an enzyme into PetroBrick expressing E. coli that results in the production of branched chain or odd chain length Fatty acids, we could extend the products produced by the PetroBrick to include branched chain or even change length alkanes. One protein whose expression has been previously shown ([[#References | [1]]]) to cause ''E. coli'' to produce branched chain and odd chain length fatty acids is FabH2, a FabH homolog from ''Bacillus subtilis''. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in even chain length linear fatty acids. However, FabH2 has been hypothesized to initiate fatty acid biosynthesis using Isobutyryl-CoA and Isovaleryl-CoA( products from Valine and Leucine degredation), resulting in 2-methyl branched fatty acid production, as well as using a 3-carbon propanoyl-CoA, resulting in odd chain length fatty acids. If we could use FabH2 and the Petrobrick to get ''E. coli'' to produce both alkanes and branched chain or odd chain length fatty acids, we should theoretically be able to produce 2-methyl branched alkanes or even chain length fatty acids.<br />
<br />
=Methods=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2 ] was constructed from oligos( refer to [https://2011.igem.org/Team:Washington/Protocols/gene_assembly protocol]). FabH2 was then amplified to add an [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 Elowitz standard RBS]. This RBS-FabH2 construct was cloned into the 3' end of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] to express [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2 as well as AAR and ADC]. In addition, we cloned fabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTG inducible vector].<br />
<br />
=FabH2 is Toxic to ''E. coli''=<br />
Throughout our experiments, we observed that cells expressing FabH2 grew significantly slower than any of our other alkane producing cells. In order to quantify this effect, we measured OD600 for alkane production constructs every 24 hours for a 72 hour period, producing a growth curve.<br />
[[File:Washington growthcurve.png|center|450px|thumb|Growth curve showing that cells overexpressing FabH2 are growth deficient. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 ADC] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 AAR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 DrR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2]<br />
]]<br />
After 24 hours, FabH2 producing cells had barely grown at all, indicating a severe growth deficiency. The cells were able to rapidly grow after 24 hours, presumably due to a mutation that counteracted the negative effects of FabH2. This toxicity could be due to branched chain fatty acid production, or due to the activity of FabH2 on straight chain substrates affecting cellular metabolism. This severe growth deficiency implies that FabH2 is being expressed, and has activity that is detrimental to cell growth. Due to FabH2's toxicity, any time that we co-expressed FabH2 and the PetroBrick on a high copy number, strong promter expression vector, total alkane yield was decimated to under 10 mg/L. Reduction of this toxicity should allow us to determine the effects of FabH2 production on alkane biosynthesis.<br />
<br />
=Reducing the Toxicity of FabH2.=<br />
We hypothesized that if we could reduce the toxicity of FabH2, we could see the production of alternative alkane products. The original FabH2 system placed the FabH2 gene under the control of a strong constitutive promoter. We thought that if we could reduce FabH2 expression, we could decrease toxicity and be able to see the effects of FabH2 production on the alkanes produced by the PetroBrick. We cloned FabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number 3k3 IPTG inducible vector]. By reducing copy number approximately 10 fold, and by having the ability to control expression, we thought that we could lessen toxicity and observe even chain length alkane production. We called this inducible, low expression construct the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590064 FabBrick], a modular add-on to the standard PetroBrick.<br />
<br />
=FabBrick Induction Results in Even Chain Length Alkane Production=<br />
The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/PetroBrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When FabH2 is induced, we see a new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that of alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinantly, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels.<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:WashingtonC16zoomed.png|middle|400px|thumb|Zoomed in spectrum. Note the parent ion at a mass of 226.]]<br />
<br />
<br />
<br />
<br />
==References==<br />
1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.<br />
<br />
2. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
3. Branched-chain fatty acid biosynthesis in Escherichia coli. Smirnova N, Reynolds KA. J Ind Microbiol Biotechnol. 2001 Oct;27(4):246-51.<br />
<br />
=Parts Submitted=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2] This part contains the FabH2 coding sequence, codon optimized for ''E. coli''<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2-ADC-AAR] This part expresses FabH2, as well as AAR and ADC, the two genes responsible for alkane production. Note that this part didn't work as expected, likely due to FabH2 toxicity. <br />
<br />
[http://partsregistry.org/Part:BBa_K590064 The FabBrick] This part expresses FabH2 in a low copynumber, inducible vector. By co-expressing this part with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick], we were able to get even chain length alkane production.</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/FabH2Team:Washington/Alkanes/Future/FabH22011-10-26T22:34:17Z<p>Hargem: /* FabBrick Induction Results in Even Chain Length Alkane Production */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
__NOTOC__<br />
<br />
<big><big><big>'''<center>Alternative Alkane Products</center>'''</big></big></big><br />
<br />
=Background=<br />
[[File:Washington_branched_CoA.png|right|450px|thumb|The branched CoA substrates used by FabH2]]<br />
The basic alkane production system is incapable of making branched chain alkanes, as ''E. coli'' normally only makes straight chain fatty acids. In addition, our basic system is only able to make odd chain length alkanes, as E. coli only normally make even chain length fatty acids. We thought that if we could introduce an enzyme into PetroBrick expressing E. coli that results in the production of branched chain or odd chain length Fatty acids, we could extend the products produced by the PetroBrick to include branched chain or even change length alkanes. One protein whose expression has been previously shown ([[#References | [1]]]) to cause ''E. coli'' to produce branched chain and odd chain length fatty acids is FabH2, a FabH homolog from ''Bacillus subtilis''. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in even chain length linear fatty acids. However, FabH2 has been hypothesized to initiate fatty acid biosynthesis using Isobutyryl-CoA and Isovaleryl-CoA( products from Valine and Leucine degredation), resulting in 2-methyl branched fatty acid production, as well as using a 3-carbon propanoyl-CoA, resulting in odd chain length fatty acids. If we could use FabH2 and the Petrobrick to get ''E. coli'' to produce both alkanes and branched chain or odd chain length fatty acids, we should theoretically be able to produce 2-methyl branched alkanes or even chain length fatty acids.<br />
<br />
=Methods=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2 ] was constructed from oligos( refer to [https://2011.igem.org/Team:Washington/Protocols/gene_assembly protocol]). FabH2 was then amplified to add an [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 Elowitz standard RBS]. This RBS-FabH2 construct was cloned into the 3' end of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] to express [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2 as well as AAR and ADC]. In addition, we cloned fabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTG inducible vector].<br />
<br />
=FabH2 is Toxic to ''E. coli''=<br />
Throughout our experiments, we observed that cells expressing FabH2 grew significantly slower than any of our other alkane producing cells. In order to quantify this effect, we measured OD600 for alkane production constructs every 24 hours for a 72 hour period, producing a growth curve.<br />
[[File:Washington growthcurve.png|center|450px|thumb|Growth curve showing that cells overexpressing FabH2 are growth deficient. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 ADC] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 AAR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 DrR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2]<br />
]]<br />
After 24 hours, FabH2 producing cells had barely grown at all, indicating a severe growth deficiency. The cells were able to rapidly grow after 24 hours, presumably due to a mutation that counteracted the negative effects of FabH2. This toxicity could be due to branched chain fatty acid production, or due to the activity of FabH2 on straight chain substrates affecting cellular metabolism. This severe growth deficiency implies that FabH2 is being expressed, and has activity that is detrimental to cell growth. Due to FabH2's toxicity, any time that we co-expressed FabH2 and the PetroBrick on a high copy number, strong promter expression vector, total alkane yield was decimated to under 10 mg/L. Reduction of this toxicity should allow us to determine the effects of FabH2 production on alkane biosynthesis.<br />
<br />
=Reducing the Toxicity of FabH2.=<br />
We hypothesized that if we could reduce the toxicity of FabH2, we could see the production of alternative alkane products. The original FabH2 system placed the FabH2 gene under the control of a strong constitutive promoter. We thought that if we could reduce FabH2 expression, we could decrease toxicity and be able to see the effects of FabH2 production on the alkanes produced by the PetroBrick. We cloned FabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number 3k3 IPTG inducible vector]. By reducing copy number approximately 10 fold, and by having the ability to control expression, we thought that we could lessen toxicity and observe even chain length alkane production. We called this inducible, low expression construct the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590064 FabBrick], a modular add-on to the standard PetroBrick.<br />
<br />
=FabBrick Induction Results in Even Chain Length Alkane Production=<br />
The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/Petrobrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When FabH2 is induced, we see a new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that of alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinantly, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels.<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
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<br />
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[[Image:WashingtonC16zoomed.png|middle|400px|thumb|Zoomed in spectrum. Note the parent ion at a mass of 226.]]<br />
<br />
<br />
<br />
<br />
==References==<br />
1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.<br />
<br />
2. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
3. Branched-chain fatty acid biosynthesis in Escherichia coli. Smirnova N, Reynolds KA. J Ind Microbiol Biotechnol. 2001 Oct;27(4):246-51.<br />
<br />
=Parts Submitted=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2] This part contains the FabH2 coding sequence, codon optimized for ''E. coli''<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2-ADC-AAR] This part expresses FabH2, as well as AAR and ADC, the two genes responsible for alkane production. Note that this part didn't work as expected, likely due to FabH2 toxicity. <br />
<br />
[http://partsregistry.org/Part:BBa_K590064 The FabBrick] This part expresses FabH2 in a low copynumber, inducible vector. By co-expressing this part with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick], we were able to get even chain length alkane production.</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/FabH2Team:Washington/Alkanes/Future/FabH22011-10-26T22:33:27Z<p>Hargem: /* FabH2 is toxic to E. coli */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
__NOTOC__<br />
<br />
<big><big><big>'''<center>Alternative Alkane Products</center>'''</big></big></big><br />
<br />
=Background=<br />
[[File:Washington_branched_CoA.png|right|450px|thumb|The branched CoA substrates used by FabH2]]<br />
The basic alkane production system is incapable of making branched chain alkanes, as ''E. coli'' normally only makes straight chain fatty acids. In addition, our basic system is only able to make odd chain length alkanes, as E. coli only normally make even chain length fatty acids. We thought that if we could introduce an enzyme into PetroBrick expressing E. coli that results in the production of branched chain or odd chain length Fatty acids, we could extend the products produced by the PetroBrick to include branched chain or even change length alkanes. One protein whose expression has been previously shown ([[#References | [1]]]) to cause ''E. coli'' to produce branched chain and odd chain length fatty acids is FabH2, a FabH homolog from ''Bacillus subtilis''. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in even chain length linear fatty acids. However, FabH2 has been hypothesized to initiate fatty acid biosynthesis using Isobutyryl-CoA and Isovaleryl-CoA( products from Valine and Leucine degredation), resulting in 2-methyl branched fatty acid production, as well as using a 3-carbon propanoyl-CoA, resulting in odd chain length fatty acids. If we could use FabH2 and the Petrobrick to get ''E. coli'' to produce both alkanes and branched chain or odd chain length fatty acids, we should theoretically be able to produce 2-methyl branched alkanes or even chain length fatty acids.<br />
<br />
=Methods=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2 ] was constructed from oligos( refer to [https://2011.igem.org/Team:Washington/Protocols/gene_assembly protocol]). FabH2 was then amplified to add an [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 Elowitz standard RBS]. This RBS-FabH2 construct was cloned into the 3' end of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] to express [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2 as well as AAR and ADC]. In addition, we cloned fabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTG inducible vector].<br />
<br />
=FabH2 is Toxic to ''E. coli''=<br />
Throughout our experiments, we observed that cells expressing FabH2 grew significantly slower than any of our other alkane producing cells. In order to quantify this effect, we measured OD600 for alkane production constructs every 24 hours for a 72 hour period, producing a growth curve.<br />
[[File:Washington growthcurve.png|center|450px|thumb|Growth curve showing that cells overexpressing FabH2 are growth deficient. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 ADC] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 AAR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 DrR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2]<br />
]]<br />
After 24 hours, FabH2 producing cells had barely grown at all, indicating a severe growth deficiency. The cells were able to rapidly grow after 24 hours, presumably due to a mutation that counteracted the negative effects of FabH2. This toxicity could be due to branched chain fatty acid production, or due to the activity of FabH2 on straight chain substrates affecting cellular metabolism. This severe growth deficiency implies that FabH2 is being expressed, and has activity that is detrimental to cell growth. Due to FabH2's toxicity, any time that we co-expressed FabH2 and the PetroBrick on a high copy number, strong promter expression vector, total alkane yield was decimated to under 10 mg/L. Reduction of this toxicity should allow us to determine the effects of FabH2 production on alkane biosynthesis.<br />
<br />
=Reducing the Toxicity of FabH2.=<br />
We hypothesized that if we could reduce the toxicity of FabH2, we could see the production of alternative alkane products. The original FabH2 system placed the FabH2 gene under the control of a strong constitutive promoter. We thought that if we could reduce FabH2 expression, we could decrease toxicity and be able to see the effects of FabH2 production on the alkanes produced by the PetroBrick. We cloned FabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number 3k3 IPTG inducible vector]. By reducing copy number approximately 10 fold, and by having the ability to control expression, we thought that we could lessen toxicity and observe even chain length alkane production. We called this inducible, low expression construct the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590064 FabBrick], a modular add-on to the standard PetroBrick.<br />
<br />
=FabBrick Induction Results in Even Chain Length Alkane Production=<br />
The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/Petrobrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When FabH2 is induced, we see a new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinantly, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels.<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]<br />
<br />
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<br />
[[Image:WashingtonC16zoomed.png|middle|400px|thumb|Zoomed in spectrum. Note the parent ion at a mass of 226.]]<br />
<br />
<br />
<br />
<br />
==References==<br />
1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.<br />
<br />
2. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
3. Branched-chain fatty acid biosynthesis in Escherichia coli. Smirnova N, Reynolds KA. J Ind Microbiol Biotechnol. 2001 Oct;27(4):246-51.<br />
<br />
=Parts Submitted=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2] This part contains the FabH2 coding sequence, codon optimized for ''E. coli''<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2-ADC-AAR] This part expresses FabH2, as well as AAR and ADC, the two genes responsible for alkane production. Note that this part didn't work as expected, likely due to FabH2 toxicity. <br />
<br />
[http://partsregistry.org/Part:BBa_K590064 The FabBrick] This part expresses FabH2 in a low copynumber, inducible vector. By co-expressing this part with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick], we were able to get even chain length alkane production.</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/FabH2Team:Washington/Alkanes/Future/FabH22011-10-26T22:32:31Z<p>Hargem: /* FabH2 is toxic to E. coli */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
__NOTOC__<br />
<br />
<big><big><big>'''<center>Alternative Alkane Products</center>'''</big></big></big><br />
<br />
=Background=<br />
[[File:Washington_branched_CoA.png|right|450px|thumb|The branched CoA substrates used by FabH2]]<br />
The basic alkane production system is incapable of making branched chain alkanes, as ''E. coli'' normally only makes straight chain fatty acids. In addition, our basic system is only able to make odd chain length alkanes, as E. coli only normally make even chain length fatty acids. We thought that if we could introduce an enzyme into PetroBrick expressing E. coli that results in the production of branched chain or odd chain length Fatty acids, we could extend the products produced by the PetroBrick to include branched chain or even change length alkanes. One protein whose expression has been previously shown ([[#References | [1]]]) to cause ''E. coli'' to produce branched chain and odd chain length fatty acids is FabH2, a FabH homolog from ''Bacillus subtilis''. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in even chain length linear fatty acids. However, FabH2 has been hypothesized to initiate fatty acid biosynthesis using Isobutyryl-CoA and Isovaleryl-CoA( products from Valine and Leucine degredation), resulting in 2-methyl branched fatty acid production, as well as using a 3-carbon propanoyl-CoA, resulting in odd chain length fatty acids. If we could use FabH2 and the Petrobrick to get ''E. coli'' to produce both alkanes and branched chain or odd chain length fatty acids, we should theoretically be able to produce 2-methyl branched alkanes or even chain length fatty acids.<br />
<br />
=Methods=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2 ] was constructed from oligos( refer to [https://2011.igem.org/Team:Washington/Protocols/gene_assembly protocol]). FabH2 was then amplified to add an [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 Elowitz standard RBS]. This RBS-FabH2 construct was cloned into the 3' end of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] to express [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2 as well as AAR and ADC]. In addition, we cloned fabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTG inducible vector].<br />
<br />
=FabH2 is toxic to ''E. coli''=<br />
Throughout our experiments, we observed that cells expressing FabH2 grew significantly slower than any of our other alkane producing cells. In order to quantify this effect, we measured OD600 for alkane production constructs every 24 hours for a 72 hour period, producing a growth curve.<br />
[[File:Washington growthcurve.png|center|450px|thumb|Growth curve showing that cells overexpressing FabH2 are growth deficient. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 ADC] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 AAR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 DrR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2]<br />
]]<br />
After 24 hours, FabH2 producing cells had barely grown at all, indicating a severe growth deficiency. The cells were able to rapidly grow after 24 hours, presumably due to a mutation that counteracted the negative effects of FabH2. This toxicity could be due to branched chain fatty acid production, or due to the activity of FabH2 on straight chain substrates affecting cellular metabolism. This severe growth deficiency implies that FabH2 is being expressed, and has activity that is detrimental to cell growth. Due to FabH2's toxicity, any time that we co-expressed FabH2 and the PetroBrick on a high copy number, strong promter expression vector, total alkane yield was decimated to under 10 mg/L. Reduction of this toxicity should allow us to determine the effects of FabH2 production on alkane biosynthesis.<br />
<br />
=Reducing the Toxicity of FabH2.=<br />
We hypothesized that if we could reduce the toxicity of FabH2, we could see the production of alternative alkane products. The original FabH2 system placed the FabH2 gene under the control of a strong constitutive promoter. We thought that if we could reduce FabH2 expression, we could decrease toxicity and be able to see the effects of FabH2 production on the alkanes produced by the PetroBrick. We cloned FabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number 3k3 IPTG inducible vector]. By reducing copy number approximately 10 fold, and by having the ability to control expression, we thought that we could lessen toxicity and observe even chain length alkane production. We called this inducible, low expression construct the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590064 FabBrick], a modular add-on to the standard PetroBrick.<br />
<br />
=FabBrick Induction Results in Even Chain Length Alkane Production=<br />
The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/Petrobrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When FabH2 is induced, we see a new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinantly, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels.<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:WashingtonC16zoomed.png|middle|400px|thumb|Zoomed in spectrum. Note the parent ion at a mass of 226.]]<br />
<br />
<br />
<br />
<br />
==References==<br />
1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.<br />
<br />
2. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
3. Branched-chain fatty acid biosynthesis in Escherichia coli. Smirnova N, Reynolds KA. J Ind Microbiol Biotechnol. 2001 Oct;27(4):246-51.<br />
<br />
=Parts Submitted=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2] This part contains the FabH2 coding sequence, codon optimized for ''E. coli''<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2-ADC-AAR] This part expresses FabH2, as well as AAR and ADC, the two genes responsible for alkane production. Note that this part didn't work as expected, likely due to FabH2 toxicity. <br />
<br />
[http://partsregistry.org/Part:BBa_K590064 The FabBrick] This part expresses FabH2 in a low copynumber, inducible vector. By co-expressing this part with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick], we were able to get even chain length alkane production.</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/FabH2Team:Washington/Alkanes/Future/FabH22011-10-26T22:28:36Z<p>Hargem: /* FabH2 is toxic to E. coli */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
__NOTOC__<br />
<br />
<big><big><big>'''<center>Alternative Alkane Products</center>'''</big></big></big><br />
<br />
=Background=<br />
[[File:Washington_branched_CoA.png|right|450px|thumb|The branched CoA substrates used by FabH2]]<br />
The basic alkane production system is incapable of making branched chain alkanes, as ''E. coli'' normally only makes straight chain fatty acids. In addition, our basic system is only able to make odd chain length alkanes, as E. coli only normally make even chain length fatty acids. We thought that if we could introduce an enzyme into PetroBrick expressing E. coli that results in the production of branched chain or odd chain length Fatty acids, we could extend the products produced by the PetroBrick to include branched chain or even change length alkanes. One protein whose expression has been previously shown ([[#References | [1]]]) to cause ''E. coli'' to produce branched chain and odd chain length fatty acids is FabH2, a FabH homolog from ''Bacillus subtilis''. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in even chain length linear fatty acids. However, FabH2 has been hypothesized to initiate fatty acid biosynthesis using Isobutyryl-CoA and Isovaleryl-CoA( products from Valine and Leucine degredation), resulting in 2-methyl branched fatty acid production, as well as using a 3-carbon propanoyl-CoA, resulting in odd chain length fatty acids. If we could use FabH2 and the Petrobrick to get ''E. coli'' to produce both alkanes and branched chain or odd chain length fatty acids, we should theoretically be able to produce 2-methyl branched alkanes or even chain length fatty acids.<br />
<br />
=Methods=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2 ] was constructed from oligos( refer to [https://2011.igem.org/Team:Washington/Protocols/gene_assembly protocol]). FabH2 was then amplified to add an [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 Elowitz standard RBS]. This RBS-FabH2 construct was cloned into the 3' end of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] to express [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2 as well as AAR and ADC]. In addition, we cloned fabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTG inducible vector].<br />
<br />
=FabH2 is toxic to ''E. coli''=<br />
Throughout our experiments, we observed that cells expressing FabH2 grew significantly slower than any of our other alkane producing cells. In order to quantify this effect, we measured OD600 for alkane production constructs every 24 hours for a 72 hour period, producing a growth curve.<br />
[[File:Washington growthcurve.png|center|450px|thumb|Growth curve showing that cells overexpressing FabH2 are growth deficient. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 ADC] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 AAR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 DrR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2]<br />
]]<br />
After 24 hours, FabH2 producing cells had barely grown at all, indicating a severe growth deficiency. The cells were able to rapidly grow after 24 hours, presumably due to a mutation that counteracted the negative effects of FabH2. This toxicity could be due to branched chain fatty acid production, or due to the activity of FabH2 on straight chain substrates affecting cellular metabolism. This severe growth deficiency implies that FabH2 is being expressed, and has activity that is detrimental to cell growth. Due to FabH2's toxicity, any time that we coexpressed fabH2 and the PetroBrick on a high copy number, strong promter expression vector, total alkane yield was decimated to under 10 mg/L. Reduction of this toxicity should allow us to determine the effects of FabH2 production on alkane biosynthesis.<br />
<br />
=Reducing the Toxicity of FabH2.=<br />
We hypothesized that if we could reduce the toxicity of FabH2, we could see the production of alternative alkane products. The original FabH2 system placed the FabH2 gene under the control of a strong constitutive promoter. We thought that if we could reduce FabH2 expression, we could decrease toxicity and be able to see the effects of FabH2 production on the alkanes produced by the PetroBrick. We cloned FabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number 3k3 IPTG inducible vector]. By reducing copy number approximately 10 fold, and by having the ability to control expression, we thought that we could lessen toxicity and observe even chain length alkane production. We called this inducible, low expression construct the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590064 FabBrick], a modular add-on to the standard PetroBrick.<br />
<br />
=FabBrick Induction Results in Even Chain Length Alkane Production=<br />
The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/Petrobrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When FabH2 is induced, we see a new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinantly, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels.<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]<br />
<br />
<br />
<br />
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<br />
<br />
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[[Image:WashingtonC16zoomed.png|middle|400px|thumb|Zoomed in spectrum. Note the parent ion at a mass of 226.]]<br />
<br />
<br />
<br />
<br />
==References==<br />
1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.<br />
<br />
2. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
3. Branched-chain fatty acid biosynthesis in Escherichia coli. Smirnova N, Reynolds KA. J Ind Microbiol Biotechnol. 2001 Oct;27(4):246-51.<br />
<br />
=Parts Submitted=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2] This part contains the FabH2 coding sequence, codon optimized for ''E. coli''<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2-ADC-AAR] This part expresses FabH2, as well as AAR and ADC, the two genes responsible for alkane production. Note that this part didn't work as expected, likely due to FabH2 toxicity. <br />
<br />
[http://partsregistry.org/Part:BBa_K590064 The FabBrick] This part expresses FabH2 in a low copynumber, inducible vector. By co-expressing this part with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick], we were able to get even chain length alkane production.</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/FabH2Team:Washington/Alkanes/Future/FabH22011-10-26T22:25:27Z<p>Hargem: /* Reducing the Toxicity of FabH2. */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
__NOTOC__<br />
<br />
<big><big><big>'''<center>Alternative Alkane Products</center>'''</big></big></big><br />
<br />
=Background=<br />
[[File:Washington_branched_CoA.png|right|450px|thumb|The branched CoA substrates used by FabH2]]<br />
The basic alkane production system is incapable of making branched chain alkanes, as ''E. coli'' normally only makes straight chain fatty acids. In addition, our basic system is only able to make odd chain length alkanes, as E. coli only normally make even chain length fatty acids. We thought that if we could introduce an enzyme into PetroBrick expressing E. coli that results in the production of branched chain or odd chain length Fatty acids, we could extend the products produced by the PetroBrick to include branched chain or even change length alkanes. One protein whose expression has been previously shown ([[#References | [1]]]) to cause ''E. coli'' to produce branched chain and odd chain length fatty acids is FabH2, a FabH homolog from ''Bacillus subtilis''. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in even chain length linear fatty acids. However, FabH2 has been hypothesized to initiate fatty acid biosynthesis using Isobutyryl-CoA and Isovaleryl-CoA( products from Valine and Leucine degredation), resulting in 2-methyl branched fatty acid production, as well as using a 3-carbon propanoyl-CoA, resulting in odd chain length fatty acids. If we could use FabH2 and the Petrobrick to get ''E. coli'' to produce both alkanes and branched chain or odd chain length fatty acids, we should theoretically be able to produce 2-methyl branched alkanes or even chain length fatty acids.<br />
<br />
=Methods=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2 ] was constructed from oligos( refer to [https://2011.igem.org/Team:Washington/Protocols/gene_assembly protocol]). FabH2 was then amplified to add an [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 Elowitz standard RBS]. This RBS-FabH2 construct was cloned into the 3' end of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] to express [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2 as well as AAR and ADC]. In addition, we cloned fabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTG inducible vector].<br />
<br />
=FabH2 is toxic to ''E. coli''=<br />
Throughout our experiments, we observed that cells expressing FabH2 grew significantly slower than any of our other alkane producing cells. In order to quantify this effect, we measured OD600 for alkane production constructs every 24 hours for a 72 hour period, producing a growth curve.<br />
[[File:Washington growthcurve.png|center|450px|thumb|Growth curve showing that cells overexpressing FabH2 are growth deficient. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 ADC] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 AAR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 DrR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2]<br />
]]<br />
After 24 hours, FabH2 producing cells had barely grown at all, indicating a severe growth deficiency. The cells were able to rapidly grow after 24 hours, presumably due to a mutation that counteracted the negative effects of FabH2. This toxicity could be due to branched chain fatty acid production, or due to the activity of FabH2 on straight chain substrates affecting cellular metabolism. This severe growth deficiency implies that FabH2 is being expressed, and has activity that is detrimental to cell growth. Due to FabH2's toxicity, any time that we coexpressed fabH2 and the PetroBrick on a high copy number, strong promter expression vector, total alkane yield was decimated. In cells expressing the normal PetroBrick, we were able to consistently Reduction of this toxicity should allow us to determine the effects of FabH2 production on alkane biosynthesis.<br />
<br />
=Reducing the Toxicity of FabH2.=<br />
We hypothesized that if we could reduce the toxicity of FabH2, we could see the production of alternative alkane products. The original FabH2 system placed the FabH2 gene under the control of a strong constitutive promoter. We thought that if we could reduce FabH2 expression, we could decrease toxicity and be able to see the effects of FabH2 production on the alkanes produced by the PetroBrick. We cloned FabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number 3k3 IPTG inducible vector]. By reducing copy number approximately 10 fold, and by having the ability to control expression, we thought that we could lessen toxicity and observe even chain length alkane production. We called this inducible, low expression construct the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590064 FabBrick], a modular add-on to the standard PetroBrick.<br />
<br />
=FabBrick Induction Results in Even Chain Length Alkane Production=<br />
The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/Petrobrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When FabH2 is induced, we see a new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinantly, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels.<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]<br />
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[[Image:WashingtonC16zoomed.png|middle|400px|thumb|Zoomed in spectrum. Note the parent ion at a mass of 226.]]<br />
<br />
<br />
<br />
<br />
==References==<br />
1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.<br />
<br />
2. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
3. Branched-chain fatty acid biosynthesis in Escherichia coli. Smirnova N, Reynolds KA. J Ind Microbiol Biotechnol. 2001 Oct;27(4):246-51.<br />
<br />
=Parts Submitted=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2] This part contains the FabH2 coding sequence, codon optimized for ''E. coli''<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2-ADC-AAR] This part expresses FabH2, as well as AAR and ADC, the two genes responsible for alkane production. Note that this part didn't work as expected, likely due to FabH2 toxicity. <br />
<br />
[http://partsregistry.org/Part:BBa_K590064 The FabBrick] This part expresses FabH2 in a low copynumber, inducible vector. By co-expressing this part with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick], we were able to get even chain length alkane production.</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/FabH2Team:Washington/Alkanes/Future/FabH22011-10-26T22:24:09Z<p>Hargem: /* Background */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
__NOTOC__<br />
<br />
<big><big><big>'''<center>Alternative Alkane Products</center>'''</big></big></big><br />
<br />
=Background=<br />
[[File:Washington_branched_CoA.png|right|450px|thumb|The branched CoA substrates used by FabH2]]<br />
The basic alkane production system is incapable of making branched chain alkanes, as ''E. coli'' normally only makes straight chain fatty acids. In addition, our basic system is only able to make odd chain length alkanes, as E. coli only normally make even chain length fatty acids. We thought that if we could introduce an enzyme into PetroBrick expressing E. coli that results in the production of branched chain or odd chain length Fatty acids, we could extend the products produced by the PetroBrick to include branched chain or even change length alkanes. One protein whose expression has been previously shown ([[#References | [1]]]) to cause ''E. coli'' to produce branched chain and odd chain length fatty acids is FabH2, a FabH homolog from ''Bacillus subtilis''. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in even chain length linear fatty acids. However, FabH2 has been hypothesized to initiate fatty acid biosynthesis using Isobutyryl-CoA and Isovaleryl-CoA( products from Valine and Leucine degredation), resulting in 2-methyl branched fatty acid production, as well as using a 3-carbon propanoyl-CoA, resulting in odd chain length fatty acids. If we could use FabH2 and the Petrobrick to get ''E. coli'' to produce both alkanes and branched chain or odd chain length fatty acids, we should theoretically be able to produce 2-methyl branched alkanes or even chain length fatty acids.<br />
<br />
=Methods=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2 ] was constructed from oligos( refer to [https://2011.igem.org/Team:Washington/Protocols/gene_assembly protocol]). FabH2 was then amplified to add an [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 Elowitz standard RBS]. This RBS-FabH2 construct was cloned into the 3' end of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] to express [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2 as well as AAR and ADC]. In addition, we cloned fabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTG inducible vector].<br />
<br />
=FabH2 is toxic to ''E. coli''=<br />
Throughout our experiments, we observed that cells expressing FabH2 grew significantly slower than any of our other alkane producing cells. In order to quantify this effect, we measured OD600 for alkane production constructs every 24 hours for a 72 hour period, producing a growth curve.<br />
[[File:Washington growthcurve.png|center|450px|thumb|Growth curve showing that cells overexpressing FabH2 are growth deficient. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 ADC] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 AAR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 DrR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2]<br />
]]<br />
After 24 hours, FabH2 producing cells had barely grown at all, indicating a severe growth deficiency. The cells were able to rapidly grow after 24 hours, presumably due to a mutation that counteracted the negative effects of FabH2. This toxicity could be due to branched chain fatty acid production, or due to the activity of FabH2 on straight chain substrates affecting cellular metabolism. This severe growth deficiency implies that FabH2 is being expressed, and has activity that is detrimental to cell growth. Due to FabH2's toxicity, any time that we coexpressed fabH2 and the PetroBrick on a high copy number, strong promter expression vector, total alkane yield was decimated. In cells expressing the normal PetroBrick, we were able to consistently Reduction of this toxicity should allow us to determine the effects of FabH2 production on alkane biosynthesis.<br />
<br />
=Reducing the Toxicity of FabH2.=<br />
We hypothesized that if we could reduce the toxicity of FabH2, we could see the production of alternative alkane products. The original FabH2 system placed the FabH2 gene under the control of a strong constitutive promoter. We thought that if we could reduce FabH2 expression, we could decrease toxicity and be able to see the effects of FabH2 production on the alkanes produced by the PetroBrick. We cloned FabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number 3k3 IPTG inducible vector]. By reducing copy number approximately 10 fold, and by having the ability to control expression, we thought that we could lessen toxicity and observe even chain length alkane production. We called this inducible, low expression construct the FabBrick, a modular add-on to the standard PetroBrick.<br />
<br />
=FabBrick Induction Results in Even Chain Length Alkane Production=<br />
The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/Petrobrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When FabH2 is induced, we see a new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinantly, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels.<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:WashingtonC16zoomed.png|middle|400px|thumb|Zoomed in spectrum. Note the parent ion at a mass of 226.]]<br />
<br />
<br />
<br />
<br />
==References==<br />
1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.<br />
<br />
2. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
3. Branched-chain fatty acid biosynthesis in Escherichia coli. Smirnova N, Reynolds KA. J Ind Microbiol Biotechnol. 2001 Oct;27(4):246-51.<br />
<br />
=Parts Submitted=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2] This part contains the FabH2 coding sequence, codon optimized for ''E. coli''<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2-ADC-AAR] This part expresses FabH2, as well as AAR and ADC, the two genes responsible for alkane production. Note that this part didn't work as expected, likely due to FabH2 toxicity. <br />
<br />
[http://partsregistry.org/Part:BBa_K590064 The FabBrick] This part expresses FabH2 in a low copynumber, inducible vector. By co-expressing this part with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick], we were able to get even chain length alkane production.</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/FabH2Team:Washington/Alkanes/Future/FabH22011-10-26T22:09:21Z<p>Hargem: /* FabBrick Induction Results in Even Chain Length Alkane Production */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
__NOTOC__<br />
<br />
<big><big><big>'''<center>Alternative Alkane Products</center>'''</big></big></big><br />
<br />
=Background=<br />
[[File:Washington_branched_CoA.png|right|450px|thumb|The branched CoA substrates used by FabH2]]<br />
The basic alkane production system is incapable of making branched chain alkanes, as ''E. coli'' normally only makes straight chain fatty acids. In addition, our basic system is only able to make odd chain length alkanes, as E. coli only normally make even chain length fatty acids. We thought that if we could introduce an enzyme into PetroBrick expressing E. coli that results in the production of branched chain or odd chain length Fatty acids, we could cause the PetroBrick to produce branched chain or even change length alkanes. One protein whose expression has been previously shown ([[#References | [1]]]) to cause ''E. coli'' to produce branched chain and odd chain length fatty acids is FabH2 from ''Bacillus subtilis''. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in even chain length linear fatty acids. However, FabH2 has been hypothesized to initiate fatty acid biosynthesis using Isobutyryl-CoA and Isovaleryl-CoA( products from Valine and Leucine degredation), resulting in 2-methyl branched fatty acid production, as well as using a 3-carbon propanoyl-CoA, resulting in odd chain length fatty acids. If we could use FabH2 and the Petrobrick to get ''E. coli'' to produce both alkanes and branched chain or odd chain length fatty acids, we should theoretically be able to produce 2-methyl branched alkanes or even chain length fatty acids.<br />
<br />
=Methods=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2 ] was constructed from oligos( refer to [https://2011.igem.org/Team:Washington/Protocols/gene_assembly protocol]). FabH2 was then amplified to add an [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 Elowitz standard RBS]. This RBS-FabH2 construct was cloned into the 3' end of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] to express [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2 as well as AAR and ADC]. In addition, we cloned fabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTG inducible vector].<br />
<br />
=FabH2 is toxic to ''E. coli''=<br />
Throughout our experiments, we observed that cells expressing FabH2 grew significantly slower than any of our other alkane producing cells. In order to quantify this effect, we measured OD600 for alkane production constructs every 24 hours for a 72 hour period, producing a growth curve.<br />
[[File:Washington growthcurve.png|center|450px|thumb|Growth curve showing that cells overexpressing FabH2 are growth deficient. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 ADC] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 AAR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 DrR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2]<br />
]]<br />
After 24 hours, FabH2 producing cells had barely grown at all, indicating a severe growth deficiency. The cells were able to rapidly grow after 24 hours, presumably due to a mutation that counteracted the negative effects of FabH2. This toxicity could be due to branched chain fatty acid production, or due to the activity of FabH2 on straight chain substrates affecting cellular metabolism. This severe growth deficiency implies that FabH2 is being expressed, and has activity that is detrimental to cell growth. Due to FabH2's toxicity, any time that we coexpressed fabH2 and the PetroBrick on a high copy number, strong promter expression vector, total alkane yield was decimated. In cells expressing the normal PetroBrick, we were able to consistently Reduction of this toxicity should allow us to determine the effects of FabH2 production on alkane biosynthesis.<br />
<br />
=Reducing the Toxicity of FabH2.=<br />
We hypothesized that if we could reduce the toxicity of FabH2, we could see the production of alternative alkane products. The original FabH2 system placed the FabH2 gene under the control of a strong constitutive promoter. We thought that if we could reduce FabH2 expression, we could decrease toxicity and be able to see the effects of FabH2 production on the alkanes produced by the PetroBrick. We cloned FabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number 3k3 IPTG inducible vector]. By reducing copy number approximately 10 fold, and by having the ability to control expression, we thought that we could lessen toxicity and observe even chain length alkane production. We called this inducible, low expression construct the FabBrick, a modular add-on to the standard PetroBrick.<br />
<br />
=FabBrick Induction Results in Even Chain Length Alkane Production=<br />
The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/Petrobrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When FabH2 is induced, we see a new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinantly, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels.<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]<br />
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[[Image:WashingtonC16zoomed.png|middle|400px|thumb|Zoomed in spectrum. Note the parent ion at a mass of 226.]]<br />
<br />
<br />
<br />
<br />
==References==<br />
1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.<br />
<br />
2. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
3. Branched-chain fatty acid biosynthesis in Escherichia coli. Smirnova N, Reynolds KA. J Ind Microbiol Biotechnol. 2001 Oct;27(4):246-51.<br />
<br />
=Parts Submitted=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2] This part contains the FabH2 coding sequence, codon optimized for ''E. coli''<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2-ADC-AAR] This part expresses FabH2, as well as AAR and ADC, the two genes responsible for alkane production. Note that this part didn't work as expected, likely due to FabH2 toxicity. <br />
<br />
[http://partsregistry.org/Part:BBa_K590064 The FabBrick] This part expresses FabH2 in a low copynumber, inducible vector. By co-expressing this part with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick], we were able to get even chain length alkane production.</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/FabH2Team:Washington/Alkanes/Future/FabH22011-10-26T22:08:34Z<p>Hargem: /* Reducing the Toxicity of FabH2. */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
__NOTOC__<br />
<br />
<big><big><big>'''<center>Alternative Alkane Products</center>'''</big></big></big><br />
<br />
=Background=<br />
[[File:Washington_branched_CoA.png|right|450px|thumb|The branched CoA substrates used by FabH2]]<br />
The basic alkane production system is incapable of making branched chain alkanes, as ''E. coli'' normally only makes straight chain fatty acids. In addition, our basic system is only able to make odd chain length alkanes, as E. coli only normally make even chain length fatty acids. We thought that if we could introduce an enzyme into PetroBrick expressing E. coli that results in the production of branched chain or odd chain length Fatty acids, we could cause the PetroBrick to produce branched chain or even change length alkanes. One protein whose expression has been previously shown ([[#References | [1]]]) to cause ''E. coli'' to produce branched chain and odd chain length fatty acids is FabH2 from ''Bacillus subtilis''. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in even chain length linear fatty acids. However, FabH2 has been hypothesized to initiate fatty acid biosynthesis using Isobutyryl-CoA and Isovaleryl-CoA( products from Valine and Leucine degredation), resulting in 2-methyl branched fatty acid production, as well as using a 3-carbon propanoyl-CoA, resulting in odd chain length fatty acids. If we could use FabH2 and the Petrobrick to get ''E. coli'' to produce both alkanes and branched chain or odd chain length fatty acids, we should theoretically be able to produce 2-methyl branched alkanes or even chain length fatty acids.<br />
<br />
=Methods=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2 ] was constructed from oligos( refer to [https://2011.igem.org/Team:Washington/Protocols/gene_assembly protocol]). FabH2 was then amplified to add an [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 Elowitz standard RBS]. This RBS-FabH2 construct was cloned into the 3' end of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] to express [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2 as well as AAR and ADC]. In addition, we cloned fabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTG inducible vector].<br />
<br />
=FabH2 is toxic to ''E. coli''=<br />
Throughout our experiments, we observed that cells expressing FabH2 grew significantly slower than any of our other alkane producing cells. In order to quantify this effect, we measured OD600 for alkane production constructs every 24 hours for a 72 hour period, producing a growth curve.<br />
[[File:Washington growthcurve.png|center|450px|thumb|Growth curve showing that cells overexpressing FabH2 are growth deficient. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 ADC] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 AAR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 DrR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2]<br />
]]<br />
After 24 hours, FabH2 producing cells had barely grown at all, indicating a severe growth deficiency. The cells were able to rapidly grow after 24 hours, presumably due to a mutation that counteracted the negative effects of FabH2. This toxicity could be due to branched chain fatty acid production, or due to the activity of FabH2 on straight chain substrates affecting cellular metabolism. This severe growth deficiency implies that FabH2 is being expressed, and has activity that is detrimental to cell growth. Due to FabH2's toxicity, any time that we coexpressed fabH2 and the PetroBrick on a high copy number, strong promter expression vector, total alkane yield was decimated. In cells expressing the normal PetroBrick, we were able to consistently Reduction of this toxicity should allow us to determine the effects of FabH2 production on alkane biosynthesis.<br />
<br />
=Reducing the Toxicity of FabH2.=<br />
We hypothesized that if we could reduce the toxicity of FabH2, we could see the production of alternative alkane products. The original FabH2 system placed the FabH2 gene under the control of a strong constitutive promoter. We thought that if we could reduce FabH2 expression, we could decrease toxicity and be able to see the effects of FabH2 production on the alkanes produced by the PetroBrick. We cloned FabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number 3k3 IPTG inducible vector]. By reducing copy number approximately 10 fold, and by having the ability to control expression, we thought that we could lessen toxicity and observe even chain length alkane production. We called this inducible, low expression construct the FabBrick, a modular add-on to the standard PetroBrick.<br />
<br />
=FabBrick Induction Results in Even Chain Length Alkane Production=<br />
The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/Petrobrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When FabH2 is induced, we see a new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinately, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels.<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]<br />
<br />
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[[Image:WashingtonC16zoomed.png|middle|400px|thumb|Zoomed in spectrum. Note the parent ion at a mass of 226.]]<br />
<br />
<br />
<br />
<br />
==References==<br />
1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.<br />
<br />
2. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
3. Branched-chain fatty acid biosynthesis in Escherichia coli. Smirnova N, Reynolds KA. J Ind Microbiol Biotechnol. 2001 Oct;27(4):246-51.<br />
<br />
=Parts Submitted=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2] This part contains the FabH2 coding sequence, codon optimized for ''E. coli''<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2-ADC-AAR] This part expresses FabH2, as well as AAR and ADC, the two genes responsible for alkane production. Note that this part didn't work as expected, likely due to FabH2 toxicity. <br />
<br />
[http://partsregistry.org/Part:BBa_K590064 The FabBrick] This part expresses FabH2 in a low copynumber, inducible vector. By co-expressing this part with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick], we were able to get even chain length alkane production.</div>Hargemhttp://2011.igem.org/File:WashingtonC16zoomed.pngFile:WashingtonC16zoomed.png2011-10-26T22:02:10Z<p>Hargem: </p>
<hr />
<div></div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/FabH2Team:Washington/Alkanes/Future/FabH22011-10-26T21:59:17Z<p>Hargem: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
__NOTOC__<br />
<br />
<big><big><big>'''<center>Alternative Alkane Products</center>'''</big></big></big><br />
<br />
=Background=<br />
[[File:Washington_branched_CoA.png|right|450px|thumb|The branched CoA substrates used by FabH2]]<br />
The basic alkane production system is incapable of making branched chain alkanes, as ''E. coli'' normally only makes straight chain fatty acids. In addition, our basic system is only able to make odd chain length alkanes, as E. coli only normally make even chain length fatty acids. We thought that if we could introduce an enzyme into PetroBrick expressing E. coli that results in the production of branched chain or odd chain length Fatty acids, we could cause the PetroBrick to produce branched chain or even change length alkanes. One protein whose expression has been previously shown ([[#References | [1]]]) to cause ''E. coli'' to produce branched chain and odd chain length fatty acids is FabH2 from ''Bacillus subtilis''. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in even chain length linear fatty acids. However, FabH2 has been hypothesized to initiate fatty acid biosynthesis using Isobutyryl-CoA and Isovaleryl-CoA( products from Valine and Leucine degredation), resulting in 2-methyl branched fatty acid production, as well as using a 3-carbon propanoyl-CoA, resulting in odd chain length fatty acids. If we could use FabH2 and the Petrobrick to get ''E. coli'' to produce both alkanes and branched chain or odd chain length fatty acids, we should theoretically be able to produce 2-methyl branched alkanes or even chain length fatty acids.<br />
<br />
=Methods=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2 ] was constructed from oligos( refer to [https://2011.igem.org/Team:Washington/Protocols/gene_assembly protocol]). FabH2 was then amplified to add an [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 Elowitz standard RBS]. This RBS-FabH2 construct was cloned into the 3' end of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] to express [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2 as well as AAR and ADC]. In addition, we cloned fabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTG inducible vector].<br />
<br />
=FabH2 is toxic to ''E. coli''=<br />
Throughout our experiments, we observed that cells expressing FabH2 grew significantly slower than any of our other alkane producing cells. In order to quantify this effect, we measured OD600 for alkane production constructs every 24 hours for a 72 hour period, producing a growth curve.<br />
[[File:Washington growthcurve.png|center|450px|thumb|Growth curve showing that cells overexpressing FabH2 are growth deficient. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 ADC] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 AAR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 DrR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2]<br />
]]<br />
After 24 hours, FabH2 producing cells had barely grown at all, indicating a severe growth deficiency. The cells were able to rapidly grow after 24 hours, presumably due to a mutation that counteracted the negative effects of FabH2. This toxicity could be due to branched chain fatty acid production, or due to the activity of FabH2 on straight chain substrates affecting cellular metabolism. This severe growth deficiency implies that FabH2 is being expressed, and has activity that is detrimental to cell growth. Due to FabH2's toxicity, any time that we coexpressed fabH2 and the PetroBrick on a high copy number, strong promter expression vector, total alkane yield was decimated. In cells expressing the normal PetroBrick, we were able to consistently Reduction of this toxicity should allow us to determine the effects of FabH2 production on alkane biosynthesis.<br />
<br />
=Reducing the Toxicity of FabH2.=<br />
We hypothesized that if we could reduce the toxicity of FabH2, we could see the production of alternative alkane products. The original FabH2 system placed the FabH2 gene under the control of a strong constituitive promoter. We thought that if we could reduce FabH2 expression, we could decrease toxicity and be able to see the effects of FabH2 production on the alkanes produced by the PetroBrick. We cloned FabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number 3k3 IPTG inducible vector]. By reducing copy number appproximetly 10 fold, and by having the ability to control expression, we thought that we could lessen toxicity and observe even chain length alkane production. We called this inducible, low expression construct the FabBrick, a modular add-on to the standard PetroBrick.<br />
=FabBrick Induction Results in Even Chain Length Alkane Production=<br />
The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/Petrobrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When FabH2 is induced, we see a new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinately, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels.<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:WashingtonC16zoomed.png|middle|400px|thumb|Zoomed in spectrum. Note the parent ion at a mass of 226.]]<br />
<br />
<br />
<br />
<br />
==References==<br />
1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.<br />
<br />
2. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
3. Branched-chain fatty acid biosynthesis in Escherichia coli. Smirnova N, Reynolds KA. J Ind Microbiol Biotechnol. 2001 Oct;27(4):246-51.<br />
<br />
=Parts Submitted=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2] This part contains the FabH2 coding sequence, codon optimized for ''E. coli''<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2-ADC-AAR] This part expresses FabH2, as well as AAR and ADC, the two genes responsible for alkane production. Note that this part didn't work as expected, likely due to FabH2 toxicity. <br />
<br />
[http://partsregistry.org/Part:BBa_K590064 The FabBrick] This part expresses FabH2 in a low copynumber, inducible vector. By co-expressing this part with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick], we were able to get even chain length alkane production.</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/FabH2Team:Washington/Alkanes/Future/FabH22011-10-26T21:50:53Z<p>Hargem: /* FabBrick Induction Results in Even Chain Length Alkane Production */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
__NOTOC__<br />
<br />
<big><big><big>'''<center>Alternative Alkane Products</center>'''</big></big></big><br />
<br />
=Background=<br />
[[File:Washington_branched_CoA.png|right|450px|thumb|The branched CoA substrates used by FabH2]]<br />
The basic alkane production system is incapable of making branched chain alkanes, as ''E. coli'' normally only makes straight chain fatty acids. In addition, our basic system is only able to make odd chain length alkanes, as E. coli only normally make even chain length fatty acids. We thought that if we could introduce an enzyme into PetroBrick expressing E. coli that results in the production of branched chain or odd chain length Fatty acids, we could cause the PetroBrick to produce branched chain or even change length alkanes. One protein whose expression has been previously shown ([[#References | [1]]]) to cause ''E. coli'' to produce branched chain and odd chain length fatty acids is FabH2 from ''Bacillus subtilis''. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in even chain length linear fatty acids. However, FabH2 has been hypothesized to initiate fatty acid biosynthesis using Isobutyryl-CoA and Isovaleryl-CoA( products from Valine and Leucine degredation), resulting in 2-methyl branched fatty acid production, as well as using a 3-carbon propanoyl-CoA, resulting in odd chain length fatty acids. If we could use FabH2 and the Petrobrick to get ''E. coli'' to produce both alkanes and branched chain or odd chain length fatty acids, we should theoretically be able to produce 2-methyl branched alkanes or even chain length fatty acids.<br />
<br />
=Methods=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2 ] was constructed from oligos( refer to [https://2011.igem.org/Team:Washington/Protocols/gene_assembly protocol]). FabH2 was then amplified to add an [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 Elowitz standard RBS]. This RBS-FabH2 construct was cloned into the 3' end of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] to express [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2 as well as AAR and ADC]. In addition, we cloned fabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTG inducible vector].<br />
<br />
=FabH2 is toxic to ''E. coli''=<br />
Throughout our experiments, we observed that cells expressing FabH2 grew significantly slower than any of our other alkane producing cells. In order to quantify this effect, we measured OD600 for alkane production constructs every 24 hours for a 72 hour period, producing a growth curve.<br />
[[File:Washington growthcurve.png|center|450px|thumb|Growth curve showing that cells overexpressing FabH2 are growth deficient. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 ADC] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 AAR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 DrR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2]<br />
]]<br />
After 24 hours, FabH2 producing cells had barely grown at all, indicating a severe growth deficiency. The cells were able to rapidly grow after 24 hours, presumably due to a mutation that counteracted the negative effects of FabH2. This toxicity could be due to branched chain fatty acid production, or due to the activity of FabH2 on straight chain substrates affecting cellular metabolism. This severe growth deficiency implies that FabH2 is being expressed, and has activity that is detrimental to cell growth. Due to FabH2's toxicity, any time that we coexpressed fabH2 and the PetroBrick on a high copy number, strong promter expression vector, total alkane yield was decimated. In cells expressing the normal PetroBrick, we were able to consistently Reduction of this toxicity should allow us to determine the effects of FabH2 production on alkane biosynthesis.<br />
<br />
=Reducing the Toxicity of FabH2.=<br />
We hypothesized that if we could reduce the toxicity of FabH2, we could see the production of alternative alkane products. The original FabH2 system placed the FabH2 gene under the control of a strong constituitive promoter. We thought that if we could reduce FabH2 expression, we could decrease toxicity and be able to see the effects of FabH2 production on the alkanes produced by the PetroBrick. We cloned FabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number 3k3 IPTG inducible vector]. By reducing copy number appproximetly 10 fold, and by having the ability to control expression, we thought that we could lessen toxicity and observe even chain length alkane production. We called this inducible, low expression construct the FabBrick, a modular add-on to the standard PetroBrick.<br />
=FabBrick Induction Results in Even Chain Length Alkane Production=<br />
The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/Petrobrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When FabH2 is induced, we see a new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinately, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels.<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]<br />
<br />
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==References==<br />
1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.<br />
<br />
2. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
3. Branched-chain fatty acid biosynthesis in Escherichia coli. Smirnova N, Reynolds KA. J Ind Microbiol Biotechnol. 2001 Oct;27(4):246-51.<br />
<br />
=Parts Submitted=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2] This part contains the FabH2 coding sequence, codon optimized for ''E. coli''<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2-ADC-AAR] This part expresses FabH2, as well as AAR and ADC, the two genes responsible for alkane production. Note that this part didn't work as expected, likely due to FabH2 toxicity. <br />
<br />
[http://partsregistry.org/Part:BBa_K590064 The FabBrick] This part expresses FabH2 in a low copynumber, inducible vector. By co-expressing this part with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick], we were able to get even chain length alkane production.</div>Hargemhttp://2011.igem.org/Team:Washington/PartsTeam:Washington/Parts2011-10-26T18:09:27Z<p>Hargem: /* Improved Parts */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Data Page'''</big></big></big></big></center><br><br><br />
<br />
<center><big>An overview of the 2011 UW iGEM team's summer projects</big></center><br />
[[File:Washington_Spacer.jpg|1px]]<br />
[[Image:UW Diesel Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Gluten Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Toolkits Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Background]]<br />
[[File:Washington_Spacer.jpg|5px]]<br />
<br />
{| style="background: white; text-align: center; width: 970px;" align="center" border="0"<br />
| '''Make It: Diesel Production'''<br />
| '''Break It: Gluten Destruction'''<br />
| '''iGEM Toolkits: Gibson Assembly<br>and Magnetosomes'''<br />
|}<br />
{| style="background: white; text-align: left; width: 965px;" border="0"<br />
|We designed and constructed a modular alkane production BioBrick, the PetroBrick, to generate alkanes, the main constituent of diesel. By expressing this BioBrick in ''E. coli'', we were able to produce alkanes of yields over 100 mg/mL.<br />
|Gluten intolerance stems from an inappropriate immune response to PQLP, the most common motif in the immunogenic peptide. We reengineered a protease, active at low pH, for strongly enhanced activity against PQLP.<br />
|We built two iGEM toolkits. The first is a set of five BioBrick vectors optimized for Gibson assembly. The second is a set of genes essential for magnetosome formation characterized in ''E. coli''.<br />
|}<br />
<br />
<br />
='''Data Summary'''=<br />
<br />
==''Data for Favorite New Parts''==<br />
<br />
<br />
==='''Diesel Production'''===<br />
<br />
:: '''1, 2.''' [http://partsregistry.org/Part:BBa_K590025 BBa_K590025: '''The PetroBrick'''] - A modular and open platform for the biological production of diesel fuel. The PetroBrick consists of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 AAR] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 ADC], each behind a standard Elowitz RBS. All of this is under regulation by a high constitutive promoter in pSB1C3.<br />
:: '''3''' [http://partsregistry.org/Part:BBa_K590064 '''The FabBrick'''] - An add-on module to the PetroBRick that causes the production of odd chain length Fatty acids. This part consists of [ http://partsregistry.org/Part:BBa_K590034 FabH2] expressed on a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTC inducible vector] These are converted into even chain length alkanes by [http://partsregistry.org/Part:BBa_K590025 BBa_K590025 the PetroBrick], completing the spectrum of linear alkane compounds that can be produced using the<br />
<br />
==='''Gluten Destruction'''===<br />
<br />
:: '''4.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590087 BBa_K590087: '''KumaMax''']- A modified version of the enzyme Kumamolisin, a protease ofthe sedolisin family native to ''Alicyclobacillus sendaiensis'' known to be active at low pH and elevated temperatures. To Kumamolisin, the mutations N291D, G319S, D358G, D368H increase activity to the PQLP peptide, an antigenic epitope in gliadin, 118-fold.<br />
<br />
==='''Gibson Assembly Toolkit'''===<br />
<br />
::'''5.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590010 BBa_K590010: '''pGA1A3'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590011 BBa_K590011: '''pGA1C3'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590012 BBa_K590012: '''pGA4C5'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590013 BBa_K590013: '''pGA4A5'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590014 BBa_K590014: '''pGA3K3'''] - These are plasmid backbones based on the bglBrick standard (BBF RFC 21) and optimized for use in Gibson assembly. These vectors are suitable replacements for the equivalent pSB vectors for iGEM teams using Gibson cloning to assemble their constructs.<br />
<br />
==='''Magnetosome Toolkit'''===<br />
<br />
:: '''6.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590015 BBa_K590015: '''sfGFP_mamK_pGA1C3'''] - This part consists of the ''mamK'' gene from ''Magnetospirillum magneticum'' strain AMB-1, as a superfolder GFP fusion, in the pGA1C3 backbone. MamK creates an actin-like filament that orients itself along the long-axis of the cell and acts as the scaffold for the alignment of magnetosome vesicles.<br />
<br />
:: '''7.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590016 BBa_K590016 '''sfGFP_mamI_pGA1C3'''] - This part consists of ''mamI'' gene from ''Magnetospirillum magneticum'' strain AMB-1, as a superfolder GFP fusion, in the pGA1C3 backbone. MamI is a membrane-localized protein required for magnetosome vesicle formation that also binds the poly-MamK filament.<br />
<br />
==''Data for Existing Parts''==<br />
<br />
::'''8.''' [http://partsregistry.org/Part:BBa_K314100:Experience K314100: '''High Constitutive Expression Cassette'''] (Washington, iGEM 2010) - We used this part to express our Petrobrick, found that it works well for expression, and entered this information in the part experience page.<br />
::'''9.''' [http://partsregistry.org/Part:pSB1A3:Experience pSB1A3] - As part of the Gibson Vector Toolkit we characterized the cloning efficiency of this plasmid backbone for Gibson assembly, using the prefix and suffix regions as primers. We found that pSB1A3 had a proper insert efficiency of 11%, compared to 99% for the equivalent Gibson-optimized pGA1A3 vector.<br />
<br />
==''Improved Parts''==<br />
<br />
::'''10.''' [http://partsregistry.org/Part:BBa_K590059 BBa_K590059], [http://partsregistry.org/Part:BBa_K590060 BBa_K590060], [http://partsregistry.org/Part:BBa_K590061 BBa_K590061: '''LuxC, D, and E'''] (Cambridge, iGEM 2010) - Formerly part of the [http://partsregistry.org/Part:BBa_K325909 LuxBrick] the genes ''luxC, D,'' and ''E'' were not separated or codon-optimized. We codon-optimized these genes and put them under the control of standard Elowitz RBS's (B0034). This was accomplished by the Alternate Aldehyde branch of the Alkane Production team.<br />
<br />
='''All Submitted Parts'''=<br />
<br />
<center><groupparts>iGEM011 Washington</groupparts></center></div>Hargemhttp://2011.igem.org/Team:Washington/PartsTeam:Washington/Parts2011-10-26T18:09:13Z<p>Hargem: /* Data for Existing Parts */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Data Page'''</big></big></big></big></center><br><br><br />
<br />
<center><big>An overview of the 2011 UW iGEM team's summer projects</big></center><br />
[[File:Washington_Spacer.jpg|1px]]<br />
[[Image:UW Diesel Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Gluten Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Toolkits Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Background]]<br />
[[File:Washington_Spacer.jpg|5px]]<br />
<br />
{| style="background: white; text-align: center; width: 970px;" align="center" border="0"<br />
| '''Make It: Diesel Production'''<br />
| '''Break It: Gluten Destruction'''<br />
| '''iGEM Toolkits: Gibson Assembly<br>and Magnetosomes'''<br />
|}<br />
{| style="background: white; text-align: left; width: 965px;" border="0"<br />
|We designed and constructed a modular alkane production BioBrick, the PetroBrick, to generate alkanes, the main constituent of diesel. By expressing this BioBrick in ''E. coli'', we were able to produce alkanes of yields over 100 mg/mL.<br />
|Gluten intolerance stems from an inappropriate immune response to PQLP, the most common motif in the immunogenic peptide. We reengineered a protease, active at low pH, for strongly enhanced activity against PQLP.<br />
|We built two iGEM toolkits. The first is a set of five BioBrick vectors optimized for Gibson assembly. The second is a set of genes essential for magnetosome formation characterized in ''E. coli''.<br />
|}<br />
<br />
<br />
='''Data Summary'''=<br />
<br />
==''Data for Favorite New Parts''==<br />
<br />
<br />
==='''Diesel Production'''===<br />
<br />
:: '''1, 2.''' [http://partsregistry.org/Part:BBa_K590025 BBa_K590025: '''The PetroBrick'''] - A modular and open platform for the biological production of diesel fuel. The PetroBrick consists of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 AAR] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 ADC], each behind a standard Elowitz RBS. All of this is under regulation by a high constitutive promoter in pSB1C3.<br />
:: '''3''' [http://partsregistry.org/Part:BBa_K590064 '''The FabBrick'''] - An add-on module to the PetroBRick that causes the production of odd chain length Fatty acids. This part consists of [ http://partsregistry.org/Part:BBa_K590034 FabH2] expressed on a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTC inducible vector] These are converted into even chain length alkanes by [http://partsregistry.org/Part:BBa_K590025 BBa_K590025 the PetroBrick], completing the spectrum of linear alkane compounds that can be produced using the<br />
<br />
==='''Gluten Destruction'''===<br />
<br />
:: '''4.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590087 BBa_K590087: '''KumaMax''']- A modified version of the enzyme Kumamolisin, a protease ofthe sedolisin family native to ''Alicyclobacillus sendaiensis'' known to be active at low pH and elevated temperatures. To Kumamolisin, the mutations N291D, G319S, D358G, D368H increase activity to the PQLP peptide, an antigenic epitope in gliadin, 118-fold.<br />
<br />
==='''Gibson Assembly Toolkit'''===<br />
<br />
::'''5.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590010 BBa_K590010: '''pGA1A3'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590011 BBa_K590011: '''pGA1C3'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590012 BBa_K590012: '''pGA4C5'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590013 BBa_K590013: '''pGA4A5'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590014 BBa_K590014: '''pGA3K3'''] - These are plasmid backbones based on the bglBrick standard (BBF RFC 21) and optimized for use in Gibson assembly. These vectors are suitable replacements for the equivalent pSB vectors for iGEM teams using Gibson cloning to assemble their constructs.<br />
<br />
==='''Magnetosome Toolkit'''===<br />
<br />
:: '''6.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590015 BBa_K590015: '''sfGFP_mamK_pGA1C3'''] - This part consists of the ''mamK'' gene from ''Magnetospirillum magneticum'' strain AMB-1, as a superfolder GFP fusion, in the pGA1C3 backbone. MamK creates an actin-like filament that orients itself along the long-axis of the cell and acts as the scaffold for the alignment of magnetosome vesicles.<br />
<br />
:: '''7.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590016 BBa_K590016 '''sfGFP_mamI_pGA1C3'''] - This part consists of ''mamI'' gene from ''Magnetospirillum magneticum'' strain AMB-1, as a superfolder GFP fusion, in the pGA1C3 backbone. MamI is a membrane-localized protein required for magnetosome vesicle formation that also binds the poly-MamK filament.<br />
<br />
==''Data for Existing Parts''==<br />
<br />
::'''8.''' [http://partsregistry.org/Part:BBa_K314100:Experience K314100: '''High Constitutive Expression Cassette'''] (Washington, iGEM 2010) - We used this part to express our Petrobrick, found that it works well for expression, and entered this information in the part experience page.<br />
::'''9.''' [http://partsregistry.org/Part:pSB1A3:Experience pSB1A3] - As part of the Gibson Vector Toolkit we characterized the cloning efficiency of this plasmid backbone for Gibson assembly, using the prefix and suffix regions as primers. We found that pSB1A3 had a proper insert efficiency of 11%, compared to 99% for the equivalent Gibson-optimized pGA1A3 vector.<br />
<br />
==''Improved Parts''==<br />
<br />
::'''9.''' [http://partsregistry.org/Part:BBa_K590059 BBa_K590059], [http://partsregistry.org/Part:BBa_K590060 BBa_K590060], [http://partsregistry.org/Part:BBa_K590061 BBa_K590061: '''LuxC, D, and E'''] (Cambridge, iGEM 2010) - Formerly part of the [http://partsregistry.org/Part:BBa_K325909 LuxBrick] the genes ''luxC, D,'' and ''E'' were not separated or codon-optimized. We codon-optimized these genes and put them under the control of standard Elowitz RBS's (B0034). This was accomplished by the Alternate Aldehyde branch of the Alkane Production team.<br />
<br />
='''All Submitted Parts'''=<br />
<br />
<center><groupparts>iGEM011 Washington</groupparts></center></div>Hargemhttp://2011.igem.org/Team:Washington/PartsTeam:Washington/Parts2011-10-26T18:06:57Z<p>Hargem: /* Magnetosome Toolkit */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Data Page'''</big></big></big></big></center><br><br><br />
<br />
<center><big>An overview of the 2011 UW iGEM team's summer projects</big></center><br />
[[File:Washington_Spacer.jpg|1px]]<br />
[[Image:UW Diesel Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Gluten Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Toolkits Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Background]]<br />
[[File:Washington_Spacer.jpg|5px]]<br />
<br />
{| style="background: white; text-align: center; width: 970px;" align="center" border="0"<br />
| '''Make It: Diesel Production'''<br />
| '''Break It: Gluten Destruction'''<br />
| '''iGEM Toolkits: Gibson Assembly<br>and Magnetosomes'''<br />
|}<br />
{| style="background: white; text-align: left; width: 965px;" border="0"<br />
|We designed and constructed a modular alkane production BioBrick, the PetroBrick, to generate alkanes, the main constituent of diesel. By expressing this BioBrick in ''E. coli'', we were able to produce alkanes of yields over 100 mg/mL.<br />
|Gluten intolerance stems from an inappropriate immune response to PQLP, the most common motif in the immunogenic peptide. We reengineered a protease, active at low pH, for strongly enhanced activity against PQLP.<br />
|We built two iGEM toolkits. The first is a set of five BioBrick vectors optimized for Gibson assembly. The second is a set of genes essential for magnetosome formation characterized in ''E. coli''.<br />
|}<br />
<br />
<br />
='''Data Summary'''=<br />
<br />
==''Data for Favorite New Parts''==<br />
<br />
<br />
==='''Diesel Production'''===<br />
<br />
:: '''1, 2.''' [http://partsregistry.org/Part:BBa_K590025 BBa_K590025: '''The PetroBrick'''] - A modular and open platform for the biological production of diesel fuel. The PetroBrick consists of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 AAR] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 ADC], each behind a standard Elowitz RBS. All of this is under regulation by a high constitutive promoter in pSB1C3.<br />
:: '''3''' [http://partsregistry.org/Part:BBa_K590064 '''The FabBrick'''] - An add-on module to the PetroBRick that causes the production of odd chain length Fatty acids. This part consists of [ http://partsregistry.org/Part:BBa_K590034 FabH2] expressed on a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTC inducible vector] These are converted into even chain length alkanes by [http://partsregistry.org/Part:BBa_K590025 BBa_K590025 the PetroBrick], completing the spectrum of linear alkane compounds that can be produced using the<br />
<br />
==='''Gluten Destruction'''===<br />
<br />
:: '''4.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590087 BBa_K590087: '''KumaMax''']- A modified version of the enzyme Kumamolisin, a protease ofthe sedolisin family native to ''Alicyclobacillus sendaiensis'' known to be active at low pH and elevated temperatures. To Kumamolisin, the mutations N291D, G319S, D358G, D368H increase activity to the PQLP peptide, an antigenic epitope in gliadin, 118-fold.<br />
<br />
==='''Gibson Assembly Toolkit'''===<br />
<br />
::'''5.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590010 BBa_K590010: '''pGA1A3'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590011 BBa_K590011: '''pGA1C3'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590012 BBa_K590012: '''pGA4C5'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590013 BBa_K590013: '''pGA4A5'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590014 BBa_K590014: '''pGA3K3'''] - These are plasmid backbones based on the bglBrick standard (BBF RFC 21) and optimized for use in Gibson assembly. These vectors are suitable replacements for the equivalent pSB vectors for iGEM teams using Gibson cloning to assemble their constructs.<br />
<br />
==='''Magnetosome Toolkit'''===<br />
<br />
:: '''6.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590015 BBa_K590015: '''sfGFP_mamK_pGA1C3'''] - This part consists of the ''mamK'' gene from ''Magnetospirillum magneticum'' strain AMB-1, as a superfolder GFP fusion, in the pGA1C3 backbone. MamK creates an actin-like filament that orients itself along the long-axis of the cell and acts as the scaffold for the alignment of magnetosome vesicles.<br />
<br />
:: '''7.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590016 BBa_K590016 '''sfGFP_mamI_pGA1C3'''] - This part consists of ''mamI'' gene from ''Magnetospirillum magneticum'' strain AMB-1, as a superfolder GFP fusion, in the pGA1C3 backbone. MamI is a membrane-localized protein required for magnetosome vesicle formation that also binds the poly-MamK filament.<br />
<br />
==''Data for Existing Parts''==<br />
<br />
::'''7.''' [http://partsregistry.org/Part:BBa_K314100:Experience K314100: '''High Constitutive Expression Cassette'''] (Washington, iGEM 2010) - We used this part to express our Petrobrick, found that it works well for expression, and entered this information in the part experience page.<br />
::'''8.''' [http://partsregistry.org/Part:pSB1A3:Experience pSB1A3] - As part of the Gibson Vector Toolkit we characterized the cloning efficiency of this plasmid backbone for Gibson assembly, using the prefix and suffix regions as primers. We found that pSB1A3 had a proper insert efficiency of 11%, compared to 99% for the equivalent Gibson-optimized pGA1A3 vector.<br />
<br />
==''Improved Parts''==<br />
<br />
::'''9.''' [http://partsregistry.org/Part:BBa_K590059 BBa_K590059], [http://partsregistry.org/Part:BBa_K590060 BBa_K590060], [http://partsregistry.org/Part:BBa_K590061 BBa_K590061: '''LuxC, D, and E'''] (Cambridge, iGEM 2010) - Formerly part of the [http://partsregistry.org/Part:BBa_K325909 LuxBrick] the genes ''luxC, D,'' and ''E'' were not separated or codon-optimized. We codon-optimized these genes and put them under the control of standard Elowitz RBS's (B0034). This was accomplished by the Alternate Aldehyde branch of the Alkane Production team.<br />
<br />
='''All Submitted Parts'''=<br />
<br />
<center><groupparts>iGEM011 Washington</groupparts></center></div>Hargemhttp://2011.igem.org/Team:Washington/PartsTeam:Washington/Parts2011-10-26T18:06:37Z<p>Hargem: /* Gibson Assembly Toolkit */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Data Page'''</big></big></big></big></center><br><br><br />
<br />
<center><big>An overview of the 2011 UW iGEM team's summer projects</big></center><br />
[[File:Washington_Spacer.jpg|1px]]<br />
[[Image:UW Diesel Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Gluten Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Toolkits Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Background]]<br />
[[File:Washington_Spacer.jpg|5px]]<br />
<br />
{| style="background: white; text-align: center; width: 970px;" align="center" border="0"<br />
| '''Make It: Diesel Production'''<br />
| '''Break It: Gluten Destruction'''<br />
| '''iGEM Toolkits: Gibson Assembly<br>and Magnetosomes'''<br />
|}<br />
{| style="background: white; text-align: left; width: 965px;" border="0"<br />
|We designed and constructed a modular alkane production BioBrick, the PetroBrick, to generate alkanes, the main constituent of diesel. By expressing this BioBrick in ''E. coli'', we were able to produce alkanes of yields over 100 mg/mL.<br />
|Gluten intolerance stems from an inappropriate immune response to PQLP, the most common motif in the immunogenic peptide. We reengineered a protease, active at low pH, for strongly enhanced activity against PQLP.<br />
|We built two iGEM toolkits. The first is a set of five BioBrick vectors optimized for Gibson assembly. The second is a set of genes essential for magnetosome formation characterized in ''E. coli''.<br />
|}<br />
<br />
<br />
='''Data Summary'''=<br />
<br />
==''Data for Favorite New Parts''==<br />
<br />
<br />
==='''Diesel Production'''===<br />
<br />
:: '''1, 2.''' [http://partsregistry.org/Part:BBa_K590025 BBa_K590025: '''The PetroBrick'''] - A modular and open platform for the biological production of diesel fuel. The PetroBrick consists of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 AAR] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 ADC], each behind a standard Elowitz RBS. All of this is under regulation by a high constitutive promoter in pSB1C3.<br />
:: '''3''' [http://partsregistry.org/Part:BBa_K590064 '''The FabBrick'''] - An add-on module to the PetroBRick that causes the production of odd chain length Fatty acids. This part consists of [ http://partsregistry.org/Part:BBa_K590034 FabH2] expressed on a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTC inducible vector] These are converted into even chain length alkanes by [http://partsregistry.org/Part:BBa_K590025 BBa_K590025 the PetroBrick], completing the spectrum of linear alkane compounds that can be produced using the<br />
<br />
==='''Gluten Destruction'''===<br />
<br />
:: '''4.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590087 BBa_K590087: '''KumaMax''']- A modified version of the enzyme Kumamolisin, a protease ofthe sedolisin family native to ''Alicyclobacillus sendaiensis'' known to be active at low pH and elevated temperatures. To Kumamolisin, the mutations N291D, G319S, D358G, D368H increase activity to the PQLP peptide, an antigenic epitope in gliadin, 118-fold.<br />
<br />
==='''Gibson Assembly Toolkit'''===<br />
<br />
::'''5.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590010 BBa_K590010: '''pGA1A3'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590011 BBa_K590011: '''pGA1C3'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590012 BBa_K590012: '''pGA4C5'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590013 BBa_K590013: '''pGA4A5'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590014 BBa_K590014: '''pGA3K3'''] - These are plasmid backbones based on the bglBrick standard (BBF RFC 21) and optimized for use in Gibson assembly. These vectors are suitable replacements for the equivalent pSB vectors for iGEM teams using Gibson cloning to assemble their constructs.<br />
<br />
==='''Magnetosome Toolkit'''===<br />
<br />
:: '''5.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590015 BBa_K590015: '''sfGFP_mamK_pGA1C3'''] - This part consists of the ''mamK'' gene from ''Magnetospirillum magneticum'' strain AMB-1, as a superfolder GFP fusion, in the pGA1C3 backbone. MamK creates an actin-like filament that orients itself along the long-axis of the cell and acts as the scaffold for the alignment of magnetosome vesicles.<br />
<br />
:: '''6.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590016 BBa_K590016 '''sfGFP_mamI_pGA1C3'''] - This part consists of ''mamI'' gene from ''Magnetospirillum magneticum'' strain AMB-1, as a superfolder GFP fusion, in the pGA1C3 backbone. MamI is a membrane-localized protein required for magnetosome vesicle formation that also binds the poly-MamK filament.<br />
<br />
==''Data for Existing Parts''==<br />
<br />
::'''7.''' [http://partsregistry.org/Part:BBa_K314100:Experience K314100: '''High Constitutive Expression Cassette'''] (Washington, iGEM 2010) - We used this part to express our Petrobrick, found that it works well for expression, and entered this information in the part experience page.<br />
::'''8.''' [http://partsregistry.org/Part:pSB1A3:Experience pSB1A3] - As part of the Gibson Vector Toolkit we characterized the cloning efficiency of this plasmid backbone for Gibson assembly, using the prefix and suffix regions as primers. We found that pSB1A3 had a proper insert efficiency of 11%, compared to 99% for the equivalent Gibson-optimized pGA1A3 vector.<br />
<br />
==''Improved Parts''==<br />
<br />
::'''9.''' [http://partsregistry.org/Part:BBa_K590059 BBa_K590059], [http://partsregistry.org/Part:BBa_K590060 BBa_K590060], [http://partsregistry.org/Part:BBa_K590061 BBa_K590061: '''LuxC, D, and E'''] (Cambridge, iGEM 2010) - Formerly part of the [http://partsregistry.org/Part:BBa_K325909 LuxBrick] the genes ''luxC, D,'' and ''E'' were not separated or codon-optimized. We codon-optimized these genes and put them under the control of standard Elowitz RBS's (B0034). This was accomplished by the Alternate Aldehyde branch of the Alkane Production team.<br />
<br />
='''All Submitted Parts'''=<br />
<br />
<center><groupparts>iGEM011 Washington</groupparts></center></div>Hargemhttp://2011.igem.org/Team:Washington/PartsTeam:Washington/Parts2011-10-26T18:06:26Z<p>Hargem: /* Gluten Destruction */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Data Page'''</big></big></big></big></center><br><br><br />
<br />
<center><big>An overview of the 2011 UW iGEM team's summer projects</big></center><br />
[[File:Washington_Spacer.jpg|1px]]<br />
[[Image:UW Diesel Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Gluten Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Toolkits Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Background]]<br />
[[File:Washington_Spacer.jpg|5px]]<br />
<br />
{| style="background: white; text-align: center; width: 970px;" align="center" border="0"<br />
| '''Make It: Diesel Production'''<br />
| '''Break It: Gluten Destruction'''<br />
| '''iGEM Toolkits: Gibson Assembly<br>and Magnetosomes'''<br />
|}<br />
{| style="background: white; text-align: left; width: 965px;" border="0"<br />
|We designed and constructed a modular alkane production BioBrick, the PetroBrick, to generate alkanes, the main constituent of diesel. By expressing this BioBrick in ''E. coli'', we were able to produce alkanes of yields over 100 mg/mL.<br />
|Gluten intolerance stems from an inappropriate immune response to PQLP, the most common motif in the immunogenic peptide. We reengineered a protease, active at low pH, for strongly enhanced activity against PQLP.<br />
|We built two iGEM toolkits. The first is a set of five BioBrick vectors optimized for Gibson assembly. The second is a set of genes essential for magnetosome formation characterized in ''E. coli''.<br />
|}<br />
<br />
<br />
='''Data Summary'''=<br />
<br />
==''Data for Favorite New Parts''==<br />
<br />
<br />
==='''Diesel Production'''===<br />
<br />
:: '''1, 2.''' [http://partsregistry.org/Part:BBa_K590025 BBa_K590025: '''The PetroBrick'''] - A modular and open platform for the biological production of diesel fuel. The PetroBrick consists of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 AAR] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 ADC], each behind a standard Elowitz RBS. All of this is under regulation by a high constitutive promoter in pSB1C3.<br />
:: '''3''' [http://partsregistry.org/Part:BBa_K590064 '''The FabBrick'''] - An add-on module to the PetroBRick that causes the production of odd chain length Fatty acids. This part consists of [ http://partsregistry.org/Part:BBa_K590034 FabH2] expressed on a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTC inducible vector] These are converted into even chain length alkanes by [http://partsregistry.org/Part:BBa_K590025 BBa_K590025 the PetroBrick], completing the spectrum of linear alkane compounds that can be produced using the<br />
<br />
==='''Gluten Destruction'''===<br />
<br />
:: '''4.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590087 BBa_K590087: '''KumaMax''']- A modified version of the enzyme Kumamolisin, a protease ofthe sedolisin family native to ''Alicyclobacillus sendaiensis'' known to be active at low pH and elevated temperatures. To Kumamolisin, the mutations N291D, G319S, D358G, D368H increase activity to the PQLP peptide, an antigenic epitope in gliadin, 118-fold.<br />
<br />
==='''Gibson Assembly Toolkit'''===<br />
<br />
::'''4.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590010 BBa_K590010: '''pGA1A3'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590011 BBa_K590011: '''pGA1C3'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590012 BBa_K590012: '''pGA4C5'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590013 BBa_K590013: '''pGA4A5'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590014 BBa_K590014: '''pGA3K3'''] - These are plasmid backbones based on the bglBrick standard (BBF RFC 21) and optimized for use in Gibson assembly. These vectors are suitable replacements for the equivalent pSB vectors for iGEM teams using Gibson cloning to assemble their constructs.<br />
<br />
==='''Magnetosome Toolkit'''===<br />
<br />
:: '''5.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590015 BBa_K590015: '''sfGFP_mamK_pGA1C3'''] - This part consists of the ''mamK'' gene from ''Magnetospirillum magneticum'' strain AMB-1, as a superfolder GFP fusion, in the pGA1C3 backbone. MamK creates an actin-like filament that orients itself along the long-axis of the cell and acts as the scaffold for the alignment of magnetosome vesicles.<br />
<br />
:: '''6.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590016 BBa_K590016 '''sfGFP_mamI_pGA1C3'''] - This part consists of ''mamI'' gene from ''Magnetospirillum magneticum'' strain AMB-1, as a superfolder GFP fusion, in the pGA1C3 backbone. MamI is a membrane-localized protein required for magnetosome vesicle formation that also binds the poly-MamK filament.<br />
<br />
==''Data for Existing Parts''==<br />
<br />
::'''7.''' [http://partsregistry.org/Part:BBa_K314100:Experience K314100: '''High Constitutive Expression Cassette'''] (Washington, iGEM 2010) - We used this part to express our Petrobrick, found that it works well for expression, and entered this information in the part experience page.<br />
::'''8.''' [http://partsregistry.org/Part:pSB1A3:Experience pSB1A3] - As part of the Gibson Vector Toolkit we characterized the cloning efficiency of this plasmid backbone for Gibson assembly, using the prefix and suffix regions as primers. We found that pSB1A3 had a proper insert efficiency of 11%, compared to 99% for the equivalent Gibson-optimized pGA1A3 vector.<br />
<br />
==''Improved Parts''==<br />
<br />
::'''9.''' [http://partsregistry.org/Part:BBa_K590059 BBa_K590059], [http://partsregistry.org/Part:BBa_K590060 BBa_K590060], [http://partsregistry.org/Part:BBa_K590061 BBa_K590061: '''LuxC, D, and E'''] (Cambridge, iGEM 2010) - Formerly part of the [http://partsregistry.org/Part:BBa_K325909 LuxBrick] the genes ''luxC, D,'' and ''E'' were not separated or codon-optimized. We codon-optimized these genes and put them under the control of standard Elowitz RBS's (B0034). This was accomplished by the Alternate Aldehyde branch of the Alkane Production team.<br />
<br />
='''All Submitted Parts'''=<br />
<br />
<center><groupparts>iGEM011 Washington</groupparts></center></div>Hargemhttp://2011.igem.org/Team:Washington/PartsTeam:Washington/Parts2011-10-26T18:05:00Z<p>Hargem: /* Diesel Production */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Data Page'''</big></big></big></big></center><br><br><br />
<br />
<center><big>An overview of the 2011 UW iGEM team's summer projects</big></center><br />
[[File:Washington_Spacer.jpg|1px]]<br />
[[Image:UW Diesel Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Gluten Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
[[File:Washington_Spacer.jpg|20px]]<br />
[[Image:UW Toolkits Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Background]]<br />
[[File:Washington_Spacer.jpg|5px]]<br />
<br />
{| style="background: white; text-align: center; width: 970px;" align="center" border="0"<br />
| '''Make It: Diesel Production'''<br />
| '''Break It: Gluten Destruction'''<br />
| '''iGEM Toolkits: Gibson Assembly<br>and Magnetosomes'''<br />
|}<br />
{| style="background: white; text-align: left; width: 965px;" border="0"<br />
|We designed and constructed a modular alkane production BioBrick, the PetroBrick, to generate alkanes, the main constituent of diesel. By expressing this BioBrick in ''E. coli'', we were able to produce alkanes of yields over 100 mg/mL.<br />
|Gluten intolerance stems from an inappropriate immune response to PQLP, the most common motif in the immunogenic peptide. We reengineered a protease, active at low pH, for strongly enhanced activity against PQLP.<br />
|We built two iGEM toolkits. The first is a set of five BioBrick vectors optimized for Gibson assembly. The second is a set of genes essential for magnetosome formation characterized in ''E. coli''.<br />
|}<br />
<br />
<br />
='''Data Summary'''=<br />
<br />
==''Data for Favorite New Parts''==<br />
<br />
<br />
==='''Diesel Production'''===<br />
<br />
:: '''1, 2.''' [http://partsregistry.org/Part:BBa_K590025 BBa_K590025: '''The PetroBrick'''] - A modular and open platform for the biological production of diesel fuel. The PetroBrick consists of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 AAR] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 ADC], each behind a standard Elowitz RBS. All of this is under regulation by a high constitutive promoter in pSB1C3.<br />
:: '''3''' [http://partsregistry.org/Part:BBa_K590064 '''The FabBrick'''] - An add-on module to the PetroBRick that causes the production of odd chain length Fatty acids. This part consists of [ http://partsregistry.org/Part:BBa_K590034 FabH2] expressed on a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTC inducible vector] These are converted into even chain length alkanes by [http://partsregistry.org/Part:BBa_K590025 BBa_K590025 the PetroBrick], completing the spectrum of linear alkane compounds that can be produced using the<br />
<br />
==='''Gluten Destruction'''===<br />
<br />
:: '''3.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590087 BBa_K590087: '''KumaMax''']- A modified version of the enzyme Kumamolisin, a protease ofthe sedolisin family native to ''Alicyclobacillus sendaiensis'' known to be active at low pH and elevated temperatures. To Kumamolisin, the mutations N291D, G319S, D358G, D368H increase activity to the PQLP peptide, an antigenic epitope in gliadin, 118-fold.<br />
<br />
==='''Gibson Assembly Toolkit'''===<br />
<br />
::'''4.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590010 BBa_K590010: '''pGA1A3'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590011 BBa_K590011: '''pGA1C3'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590012 BBa_K590012: '''pGA4C5'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590013 BBa_K590013: '''pGA4A5'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590014 BBa_K590014: '''pGA3K3'''] - These are plasmid backbones based on the bglBrick standard (BBF RFC 21) and optimized for use in Gibson assembly. These vectors are suitable replacements for the equivalent pSB vectors for iGEM teams using Gibson cloning to assemble their constructs.<br />
<br />
==='''Magnetosome Toolkit'''===<br />
<br />
:: '''5.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590015 BBa_K590015: '''sfGFP_mamK_pGA1C3'''] - This part consists of the ''mamK'' gene from ''Magnetospirillum magneticum'' strain AMB-1, as a superfolder GFP fusion, in the pGA1C3 backbone. MamK creates an actin-like filament that orients itself along the long-axis of the cell and acts as the scaffold for the alignment of magnetosome vesicles.<br />
<br />
:: '''6.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590016 BBa_K590016 '''sfGFP_mamI_pGA1C3'''] - This part consists of ''mamI'' gene from ''Magnetospirillum magneticum'' strain AMB-1, as a superfolder GFP fusion, in the pGA1C3 backbone. MamI is a membrane-localized protein required for magnetosome vesicle formation that also binds the poly-MamK filament.<br />
<br />
==''Data for Existing Parts''==<br />
<br />
::'''7.''' [http://partsregistry.org/Part:BBa_K314100:Experience K314100: '''High Constitutive Expression Cassette'''] (Washington, iGEM 2010) - We used this part to express our Petrobrick, found that it works well for expression, and entered this information in the part experience page.<br />
::'''8.''' [http://partsregistry.org/Part:pSB1A3:Experience pSB1A3] - As part of the Gibson Vector Toolkit we characterized the cloning efficiency of this plasmid backbone for Gibson assembly, using the prefix and suffix regions as primers. We found that pSB1A3 had a proper insert efficiency of 11%, compared to 99% for the equivalent Gibson-optimized pGA1A3 vector.<br />
<br />
==''Improved Parts''==<br />
<br />
::'''9.''' [http://partsregistry.org/Part:BBa_K590059 BBa_K590059], [http://partsregistry.org/Part:BBa_K590060 BBa_K590060], [http://partsregistry.org/Part:BBa_K590061 BBa_K590061: '''LuxC, D, and E'''] (Cambridge, iGEM 2010) - Formerly part of the [http://partsregistry.org/Part:BBa_K325909 LuxBrick] the genes ''luxC, D,'' and ''E'' were not separated or codon-optimized. We codon-optimized these genes and put them under the control of standard Elowitz RBS's (B0034). This was accomplished by the Alternate Aldehyde branch of the Alkane Production team.<br />
<br />
='''All Submitted Parts'''=<br />
<br />
<center><groupparts>iGEM011 Washington</groupparts></center></div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/FabH2Team:Washington/Alkanes/Future/FabH22011-10-26T17:58:09Z<p>Hargem: /* FabH2 is toxic to E. coli */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
__NOTOC__<br />
<br />
<big><big><big>'''<center>Alternative Alkane Products</center>'''</big></big></big><br />
<br />
=Background=<br />
[[File:Washington_branched_CoA.png|right|450px|thumb|The branched CoA substrates used by FabH2]]<br />
The basic alkane production system is incapable of making branched chain alkanes, as ''E. coli'' normally only makes straight chain fatty acids. In addition, our basic system is only able to make odd chain length alkanes, as E. coli only normally make even chain length fatty acids. We thought that if we could introduce an enzyme into PetroBrick expressing E. coli that results in the production of branched chain or odd chain length Fatty acids, we could cause the PetroBrick to produce branched chain or even change length alkanes. One protein whose expression has been previously shown ([[#References | [1]]]) to cause ''E. coli'' to produce branched chain and odd chain length fatty acids is FabH2 from ''Bacillus subtilis''. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in even chain length linear fatty acids. However, FabH2 has been hypothesized to initiate fatty acid biosynthesis using Isobutyryl-CoA and Isovaleryl-CoA( products from Valine and Leucine degredation), resulting in 2-methyl branched fatty acid production, as well as using a 3-carbon propanoyl-CoA, resulting in odd chain length fatty acids. If we could use FabH2 and the Petrobrick to get ''E. coli'' to produce both alkanes and branched chain or odd chain length fatty acids, we should theoretically be able to produce 2-methyl branched alkanes or even chain length fatty acids.<br />
<br />
=Methods=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2 ] was constructed from oligos( refer to [https://2011.igem.org/Team:Washington/Protocols/gene_assembly protocol]). FabH2 was then amplified to add an [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 Elowitz standard RBS]. This RBS-FabH2 construct was cloned into the 3' end of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] to express [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2 as well as AAR and ADC]. In addition, we cloned fabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTG inducible vector].<br />
<br />
=FabH2 is toxic to ''E. coli''=<br />
Throughout our experiments, we observed that cells expressing FabH2 grew significantly slower than any of our other alkane producing cells. In order to quantify this effect, we measured OD600 for alkane production constructs every 24 hours for a 72 hour period, producing a growth curve.<br />
[[File:Washington growthcurve.png|center|450px|thumb|Growth curve showing that cells overexpressing FabH2 are growth deficient. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 ADC] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 AAR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 DrR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2]<br />
]]<br />
After 24 hours, FabH2 producing cells had barely grown at all, indicating a severe growth deficiency. The cells were able to rapidly grow after 24 hours, presumably due to a mutation that counteracted the negative effects of FabH2. This toxicity could be due to branched chain fatty acid production, or due to the activity of FabH2 on straight chain substrates affecting cellular metabolism. This severe growth deficiency implies that FabH2 is being expressed, and has activity that is detrimental to cell growth. Due to FabH2's toxicity, any time that we coexpressed fabH2 and the PetroBrick on a high copy number, strong promter expression vector, total alkane yield was decimated. In cells expressing the normal PetroBrick, we were able to consistently Reduction of this toxicity should allow us to determine the effects of FabH2 production on alkane biosynthesis.<br />
<br />
=Reducing the Toxicity of FabH2.=<br />
We hypothesized that if we could reduce the toxicity of FabH2, we could see the production of alternative alkane products. The original FabH2 system placed the FabH2 gene under the control of a strong constituitive promoter. We thought that if we could reduce FabH2 expression, we could decrease toxicity and be able to see the effects of FabH2 production on the alkanes produced by the PetroBrick. We cloned FabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number 3k3 IPTG inducible vector]. By reducing copy number appproximetly 10 fold, and by having the ability to control expression, we thought that we could lessen toxicity and observe even chain length alkane production. We called this inducible, low expression construct the FabBrick, a modular add-on to the standard PetroBrick.<br />
=FabBrick Induction Results in Even Chain Length Alkane Production=<br />
The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/Petrobrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When FabH2 is induced, we see a strong new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinately, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels.<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
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[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]<br />
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==References==<br />
1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.<br />
<br />
2. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
3. Branched-chain fatty acid biosynthesis in Escherichia coli. Smirnova N, Reynolds KA. J Ind Microbiol Biotechnol. 2001 Oct;27(4):246-51.<br />
<br />
=Parts Submitted=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2] This part contains the FabH2 coding sequence, codon optimized for ''E. coli''<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2-ADC-AAR] This part expresses FabH2, as well as AAR and ADC, the two genes responsible for alkane production. Note that this part didn't work as expected, likely due to FabH2 toxicity. <br />
<br />
[http://partsregistry.org/Part:BBa_K590064 The FabBrick] This part expresses FabH2 in a low copynumber, inducible vector. By co-expressing this part with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick], we were able to get even chain length alkane production.</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/FabH2Team:Washington/Alkanes/Future/FabH22011-10-26T17:45:28Z<p>Hargem: /* FabBrick Induction Results in Even Chain Length Alkane Production */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
__NOTOC__<br />
<br />
<big><big><big>'''<center>Alternative Alkane Products</center>'''</big></big></big><br />
<br />
=Background=<br />
[[File:Washington_branched_CoA.png|right|450px|thumb|The branched CoA substrates used by FabH2]]<br />
The basic alkane production system is incapable of making branched chain alkanes, as ''E. coli'' normally only makes straight chain fatty acids. In addition, our basic system is only able to make odd chain length alkanes, as E. coli only normally make even chain length fatty acids. We thought that if we could introduce an enzyme into PetroBrick expressing E. coli that results in the production of branched chain or odd chain length Fatty acids, we could cause the PetroBrick to produce branched chain or even change length alkanes. One protein whose expression has been previously shown ([[#References | [1]]]) to cause ''E. coli'' to produce branched chain and odd chain length fatty acids is FabH2 from ''Bacillus subtilis''. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in even chain length linear fatty acids. However, FabH2 has been hypothesized to initiate fatty acid biosynthesis using Isobutyryl-CoA and Isovaleryl-CoA( products from Valine and Leucine degredation), resulting in 2-methyl branched fatty acid production, as well as using a 3-carbon propanoyl-CoA, resulting in odd chain length fatty acids. If we could use FabH2 and the Petrobrick to get ''E. coli'' to produce both alkanes and branched chain or odd chain length fatty acids, we should theoretically be able to produce 2-methyl branched alkanes or even chain length fatty acids.<br />
<br />
=Methods=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2 ] was constructed from oligos( refer to [https://2011.igem.org/Team:Washington/Protocols/gene_assembly protocol]). FabH2 was then amplified to add an [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 Elowitz standard RBS]. This RBS-FabH2 construct was cloned into the 3' end of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] to express [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2 as well as AAR and ADC]. In addition, we cloned fabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTG inducible vector].<br />
<br />
=FabH2 is toxic to ''E. coli''=<br />
Throughout our experiments, we observed that cells expressing FabH2 grew significantly slower than any of our other alkane producing cells. In order to quantify this effect, we measured OD600 for alkane production constructs every 24 hours for a 72 hour period, producing a growth curve.<br />
[[File:Washington growthcurve.png|center|450px|thumb|Growth curve showing that cells expressing FabH2 are growth deficient. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 ADC] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 AAR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 DrR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2]<br />
]]<br />
After 24 hours, FabH2 producing cells had barely grown at all, indicating a severe growth deficiency. The cells were able to rapidly grow after 24 hours, presumably due to a mutation that counteracted the negative effects of FabH2. This toxicity could be due to branched chain fatty acid production, or due to the activity of FabH2 on straight chain substrates affecting cellular metabolism. This severe growth deficiency implies that FabH2 is being expressed, and has activity that is detrimental to cell growth. Due to FabH2's toxicity, any time that we coexpressed fabH2 and the PetroBrick on a high copy number, strong promter expression vector, total alkane yield was decimated. In cells expressing the normal PetroBrick, we were able to consistently Reduction of this toxicity should allow us to determine the effects of FabH2 production on alkane biosynthesis.<br />
<br />
=Reducing the Toxicity of FabH2.=<br />
We hypothesized that if we could reduce the toxicity of FabH2, we could see the production of alternative alkane products. The original FabH2 system placed the FabH2 gene under the control of a strong constituitive promoter. We thought that if we could reduce FabH2 expression, we could decrease toxicity and be able to see the effects of FabH2 production on the alkanes produced by the PetroBrick. We cloned FabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number 3k3 IPTG inducible vector]. By reducing copy number appproximetly 10 fold, and by having the ability to control expression, we thought that we could lessen toxicity and observe even chain length alkane production. We called this inducible, low expression construct the FabBrick, a modular add-on to the standard PetroBrick.<br />
=FabBrick Induction Results in Even Chain Length Alkane Production=<br />
The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/Petrobrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When FabH2 is induced, we see a strong new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinately, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels.<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
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[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]<br />
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==References==<br />
1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.<br />
<br />
2. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
3. Branched-chain fatty acid biosynthesis in Escherichia coli. Smirnova N, Reynolds KA. J Ind Microbiol Biotechnol. 2001 Oct;27(4):246-51.<br />
<br />
=Parts Submitted=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2] This part contains the FabH2 coding sequence, codon optimized for ''E. coli''<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2-ADC-AAR] This part expresses FabH2, as well as AAR and ADC, the two genes responsible for alkane production. Note that this part didn't work as expected, likely due to FabH2 toxicity. <br />
<br />
[http://partsregistry.org/Part:BBa_K590064 The FabBrick] This part expresses FabH2 in a low copynumber, inducible vector. By co-expressing this part with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick], we were able to get even chain length alkane production.</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/FabH2Team:Washington/Alkanes/Future/FabH22011-10-26T17:43:37Z<p>Hargem: /* Parts Submitted */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
__NOTOC__<br />
<br />
<big><big><big>'''<center>Alternative Alkane Products</center>'''</big></big></big><br />
<br />
=Background=<br />
[[File:Washington_branched_CoA.png|right|450px|thumb|The branched CoA substrates used by FabH2]]<br />
The basic alkane production system is incapable of making branched chain alkanes, as ''E. coli'' normally only makes straight chain fatty acids. In addition, our basic system is only able to make odd chain length alkanes, as E. coli only normally make even chain length fatty acids. We thought that if we could introduce an enzyme into PetroBrick expressing E. coli that results in the production of branched chain or odd chain length Fatty acids, we could cause the PetroBrick to produce branched chain or even change length alkanes. One protein whose expression has been previously shown ([[#References | [1]]]) to cause ''E. coli'' to produce branched chain and odd chain length fatty acids is FabH2 from ''Bacillus subtilis''. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in even chain length linear fatty acids. However, FabH2 has been hypothesized to initiate fatty acid biosynthesis using Isobutyryl-CoA and Isovaleryl-CoA( products from Valine and Leucine degredation), resulting in 2-methyl branched fatty acid production, as well as using a 3-carbon propanoyl-CoA, resulting in odd chain length fatty acids. If we could use FabH2 and the Petrobrick to get ''E. coli'' to produce both alkanes and branched chain or odd chain length fatty acids, we should theoretically be able to produce 2-methyl branched alkanes or even chain length fatty acids.<br />
<br />
=Methods=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2 ] was constructed from oligos( refer to [https://2011.igem.org/Team:Washington/Protocols/gene_assembly protocol]). FabH2 was then amplified to add an [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 Elowitz standard RBS]. This RBS-FabH2 construct was cloned into the 3' end of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] to express [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2 as well as AAR and ADC]. In addition, we cloned fabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTG inducible vector].<br />
<br />
=FabH2 is toxic to ''E. coli''=<br />
Throughout our experiments, we observed that cells expressing FabH2 grew significantly slower than any of our other alkane producing cells. In order to quantify this effect, we measured OD600 for alkane production constructs every 24 hours for a 72 hour period, producing a growth curve.<br />
[[File:Washington growthcurve.png|center|450px|thumb|Growth curve showing that cells expressing FabH2 are growth deficient. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 ADC] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 AAR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 DrR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2]<br />
]]<br />
After 24 hours, FabH2 producing cells had barely grown at all, indicating a severe growth deficiency. The cells were able to rapidly grow after 24 hours, presumably due to a mutation that counteracted the negative effects of FabH2. This toxicity could be due to branched chain fatty acid production, or due to the activity of FabH2 on straight chain substrates affecting cellular metabolism. This severe growth deficiency implies that FabH2 is being expressed, and has activity that is detrimental to cell growth. Due to FabH2's toxicity, any time that we coexpressed fabH2 and the PetroBrick on a high copy number, strong promter expression vector, total alkane yield was decimated. In cells expressing the normal PetroBrick, we were able to consistently Reduction of this toxicity should allow us to determine the effects of FabH2 production on alkane biosynthesis.<br />
<br />
=Reducing the Toxicity of FabH2.=<br />
We hypothesized that if we could reduce the toxicity of FabH2, we could see the production of alternative alkane products. The original FabH2 system placed the FabH2 gene under the control of a strong constituitive promoter. We thought that if we could reduce FabH2 expression, we could decrease toxicity and be able to see the effects of FabH2 production on the alkanes produced by the PetroBrick. We cloned FabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number 3k3 IPTG inducible vector]. By reducing copy number appproximetly 10 fold, and by having the ability to control expression, we thought that we could lessen toxicity and observe even chain length alkane production. We called this inducible, low expression construct the FabBrick, a modular add-on to the standard PetroBrick.<br />
=FabBrick Induction Results in Even Chain Length Alkane Production=<br />
The FabBrick was co-transformed with the Petrobrick in XL1-Blue E. coli. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/Petrobrick cultures, and on cells expressing only the PetroBrick.<br />
<br />
There was no significant difference between the GC peaks in the PetroBrick extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When FabH2 is induced, we see a strong new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinately, and expands the PetroBrick system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels.<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
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[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]<br />
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==References==<br />
1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.<br />
<br />
2. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
3. Branched-chain fatty acid biosynthesis in Escherichia coli. Smirnova N, Reynolds KA. J Ind Microbiol Biotechnol. 2001 Oct;27(4):246-51.<br />
<br />
=Parts Submitted=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2] This part contains the FabH2 coding sequence, codon optimized for ''E. coli''<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2-ADC-AAR] This part expresses FabH2, as well as AAR and ADC, the two genes responsible for alkane production. Note that this part didn't work as expected, likely due to FabH2 toxicity. <br />
<br />
[http://partsregistry.org/Part:BBa_K590064 The FabBrick] This part expresses FabH2 in a low copynumber, inducible vector. By co-expressing this part with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick], we were able to get even chain length alkane production.</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/FabH2Team:Washington/Alkanes/Future/FabH22011-10-26T17:40:45Z<p>Hargem: /* Parts Submitted */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
__NOTOC__<br />
<br />
<big><big><big>'''<center>Alternative Alkane Products</center>'''</big></big></big><br />
<br />
=Background=<br />
[[File:Washington_branched_CoA.png|right|450px|thumb|The branched CoA substrates used by FabH2]]<br />
The basic alkane production system is incapable of making branched chain alkanes, as ''E. coli'' normally only makes straight chain fatty acids. In addition, our basic system is only able to make odd chain length alkanes, as E. coli only normally make even chain length fatty acids. We thought that if we could introduce an enzyme into PetroBrick expressing E. coli that results in the production of branched chain or odd chain length Fatty acids, we could cause the PetroBrick to produce branched chain or even change length alkanes. One protein whose expression has been previously shown ([[#References | [1]]]) to cause ''E. coli'' to produce branched chain and odd chain length fatty acids is FabH2 from ''Bacillus subtilis''. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in even chain length linear fatty acids. However, FabH2 has been hypothesized to initiate fatty acid biosynthesis using Isobutyryl-CoA and Isovaleryl-CoA( products from Valine and Leucine degredation), resulting in 2-methyl branched fatty acid production, as well as using a 3-carbon propanoyl-CoA, resulting in odd chain length fatty acids. If we could use FabH2 and the Petrobrick to get ''E. coli'' to produce both alkanes and branched chain or odd chain length fatty acids, we should theoretically be able to produce 2-methyl branched alkanes or even chain length fatty acids.<br />
<br />
=Methods=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2 ] was constructed from oligos( refer to [https://2011.igem.org/Team:Washington/Protocols/gene_assembly protocol]). FabH2 was then amplified to add an [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 Elowitz standard RBS]. This RBS-FabH2 construct was cloned into the 3' end of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] to express [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2 as well as AAR and ADC]. In addition, we cloned fabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTG inducible vector].<br />
<br />
=FabH2 is toxic to ''E. coli''=<br />
Throughout our experiments, we observed that cells expressing FabH2 grew significantly slower than any of our other alkane producing cells. In order to quantify this effect, we measured OD600 for alkane production constructs every 24 hours for a 72 hour period, producing a growth curve.<br />
[[File:Washington growthcurve.png|center|450px|thumb|Growth curve showing that cells expressing FabH2 are growth deficient. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 ADC] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 AAR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 DrR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2]<br />
]]<br />
After 24 hours, FabH2 producing cells had barely grown at all, indicating a severe growth deficiency. The cells were able to rapidly grow after 24 hours, presumably due to a mutation that counteracted the negative effects of FabH2. This toxicity could be due to branched chain fatty acid production, or due to the activity of FabH2 on straight chain substrates affecting cellular metabolism. This severe growth deficiency implies that FabH2 is being expressed, and has activity that is detrimental to cell growth. Due to FabH2's toxicity, any time that we coexpressed fabH2 and the PetroBrick on a high copy number, strong promter expression vector, total alkane yield was decimated. In cells expressing the normal PetroBrick, we were able to consistently Reduction of this toxicity should allow us to determine the effects of FabH2 production on alkane biosynthesis.<br />
<br />
=Reducing the Toxicity of FabH2.=<br />
We hypothesized that if we could reduce the toxicity of FabH2, we could see the production of alternative alkane products. The original FabH2 system placed the FabH2 gene under the control of a strong constituitive promoter. We thought that if we could reduce FabH2 expression, we could decrease toxicity and be able to see the effects of FabH2 production on the alkanes produced by the PetroBrick. We cloned FabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number 3k3 IPTG inducible vector]. By reducing copy number appproximetly 10 fold, and by having the ability to control expression, we thought that we could lessen toxicity and observe even chain length alkane production. We called this inducible, low expression construct the FabBrick, a modular add-on to the standard PetroBrick.<br />
=FabBrick Induction Results in Even Chain Length Alkane Production=<br />
The FabBrick was co-transformed with the Petrobrick in XL1-Blue E. coli. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/Petrobrick cultures, and on cells expressing only the PetroBrick.<br />
<br />
There was no significant difference between the GC peaks in the PetroBrick extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When FabH2 is induced, we see a strong new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinately, and expands the PetroBrick system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels.<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]<br />
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==References==<br />
1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.<br />
<br />
2. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
3. Branched-chain fatty acid biosynthesis in Escherichia coli. Smirnova N, Reynolds KA. J Ind Microbiol Biotechnol. 2001 Oct;27(4):246-51.<br />
<br />
=Parts Submitted=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2] This part contains the FabH2 coding sequence, codon optimized for ''E. coli''<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2-ADC-AAR] This part expresses FabH2, as well as AAR and ADC, the two genes responsible for alkane production. Note that this part didn't work as expected, likely due to FabH2 toxicity. <br />
[http://partsregistry.org/Part:BBa_K590064 The FabBrick] This part expresses FabH2 in a low copynumber, inducible vector. By co-expressing this part with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the Petrobrick], we were able to get even chain length alkane production.</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/FabH2Team:Washington/Alkanes/Future/FabH22011-10-26T17:35:50Z<p>Hargem: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
__NOTOC__<br />
<br />
<big><big><big>'''<center>Alternative Alkane Products</center>'''</big></big></big><br />
<br />
=Background=<br />
[[File:Washington_branched_CoA.png|right|450px|thumb|The branched CoA substrates used by FabH2]]<br />
The basic alkane production system is incapable of making branched chain alkanes, as ''E. coli'' normally only makes straight chain fatty acids. In addition, our basic system is only able to make odd chain length alkanes, as E. coli only normally make even chain length fatty acids. We thought that if we could introduce an enzyme into PetroBrick expressing E. coli that results in the production of branched chain or odd chain length Fatty acids, we could cause the PetroBrick to produce branched chain or even change length alkanes. One protein whose expression has been previously shown ([[#References | [1]]]) to cause ''E. coli'' to produce branched chain and odd chain length fatty acids is FabH2 from ''Bacillus subtilis''. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in even chain length linear fatty acids. However, FabH2 has been hypothesized to initiate fatty acid biosynthesis using Isobutyryl-CoA and Isovaleryl-CoA( products from Valine and Leucine degredation), resulting in 2-methyl branched fatty acid production, as well as using a 3-carbon propanoyl-CoA, resulting in odd chain length fatty acids. If we could use FabH2 and the Petrobrick to get ''E. coli'' to produce both alkanes and branched chain or odd chain length fatty acids, we should theoretically be able to produce 2-methyl branched alkanes or even chain length fatty acids.<br />
<br />
=Methods=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2 ] was constructed from oligos( refer to [https://2011.igem.org/Team:Washington/Protocols/gene_assembly protocol]). FabH2 was then amplified to add an [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 Elowitz standard RBS]. This RBS-FabH2 construct was cloned into the 3' end of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] to express [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2 as well as AAR and ADC]. In addition, we cloned fabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTG inducible vector].<br />
<br />
=FabH2 is toxic to ''E. coli''=<br />
Throughout our experiments, we observed that cells expressing FabH2 grew significantly slower than any of our other alkane producing cells. In order to quantify this effect, we measured OD600 for alkane production constructs every 24 hours for a 72 hour period, producing a growth curve.<br />
[[File:Washington growthcurve.png|center|450px|thumb|Growth curve showing that cells expressing FabH2 are growth deficient. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 ADC] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 AAR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 DrR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2]<br />
]]<br />
After 24 hours, FabH2 producing cells had barely grown at all, indicating a severe growth deficiency. The cells were able to rapidly grow after 24 hours, presumably due to a mutation that counteracted the negative effects of FabH2. This toxicity could be due to branched chain fatty acid production, or due to the activity of FabH2 on straight chain substrates affecting cellular metabolism. This severe growth deficiency implies that FabH2 is being expressed, and has activity that is detrimental to cell growth. Due to FabH2's toxicity, any time that we coexpressed fabH2 and the PetroBrick on a high copy number, strong promter expression vector, total alkane yield was decimated. In cells expressing the normal PetroBrick, we were able to consistently Reduction of this toxicity should allow us to determine the effects of FabH2 production on alkane biosynthesis.<br />
<br />
=Reducing the Toxicity of FabH2.=<br />
We hypothesized that if we could reduce the toxicity of FabH2, we could see the production of alternative alkane products. The original FabH2 system placed the FabH2 gene under the control of a strong constituitive promoter. We thought that if we could reduce FabH2 expression, we could decrease toxicity and be able to see the effects of FabH2 production on the alkanes produced by the PetroBrick. We cloned FabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number 3k3 IPTG inducible vector]. By reducing copy number appproximetly 10 fold, and by having the ability to control expression, we thought that we could lessen toxicity and observe even chain length alkane production. We called this inducible, low expression construct the FabBrick, a modular add-on to the standard PetroBrick.<br />
=FabBrick Induction Results in Even Chain Length Alkane Production=<br />
The FabBrick was co-transformed with the Petrobrick in XL1-Blue E. coli. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/Petrobrick cultures, and on cells expressing only the PetroBrick.<br />
<br />
There was no significant difference between the GC peaks in the PetroBrick extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When FabH2 is induced, we see a strong new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinately, and expands the PetroBrick system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels.<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
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[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]<br />
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==References==<br />
1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.<br />
<br />
2. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
3. Branched-chain fatty acid biosynthesis in Escherichia coli. Smirnova N, Reynolds KA. J Ind Microbiol Biotechnol. 2001 Oct;27(4):246-51.<br />
<br />
=Parts Submitted=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2] This part contains the FabH2 coding sequence, codon optimized for ''E. coli''<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2-ADC-AAR] This part expresses FabH2, as well as AAR and ADC, the two genes responsible for alkane production.</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/FabH2Team:Washington/Alkanes/Future/FabH22011-10-26T17:35:35Z<p>Hargem: /* FabBrick Induction Results in Even Chain Length Alkane Production */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
__NOTOC__<br />
<br />
<big><big><big>'''<center>Alternative Alkane Products</center>'''</big></big></big><br />
<br />
=Background=<br />
[[File:Washington_branched_CoA.png|right|450px|thumb|The branched CoA substrates used by FabH2]]<br />
The basic alkane production system is incapable of making branched chain alkanes, as ''E. coli'' normally only makes straight chain fatty acids. In addition, our basic system is only able to make odd chain length alkanes, as E. coli only normally make even chain length fatty acids. We thought that if we could introduce an enzyme into PetroBrick expressing E. coli that results in the production of branched chain or odd chain length Fatty acids, we could cause the PetroBrick to produce branched chain or even change length alkanes. One protein whose expression has been previously shown ([[#References | [1]]]) to cause ''E. coli'' to produce branched chain and odd chain length fatty acids is FabH2 from ''Bacillus subtilis''. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in even chain length linear fatty acids. However, FabH2 has been hypothesized to initiate fatty acid biosynthesis using Isobutyryl-CoA and Isovaleryl-CoA( products from Valine and Leucine degredation), resulting in 2-methyl branched fatty acid production, as well as using a 3-carbon propanoyl-CoA, resulting in odd chain length fatty acids. If we could use FabH2 and the Petrobrick to get ''E. coli'' to produce both alkanes and branched chain or odd chain length fatty acids, we should theoretically be able to produce 2-methyl branched alkanes or even chain length fatty acids.<br />
<br />
=Methods=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2 ] was constructed from oligos( refer to [https://2011.igem.org/Team:Washington/Protocols/gene_assembly protocol]). FabH2 was then amplified to add an [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 Elowitz standard RBS]. This RBS-FabH2 construct was cloned into the 3' end of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] to express [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2 as well as AAR and ADC]. In addition, we cloned fabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTG inducible vector].<br />
<br />
=FabH2 is toxic to ''E. coli''=<br />
Throughout our experiments, we observed that cells expressing FabH2 grew significantly slower than any of our other alkane producing cells. In order to quantify this effect, we measured OD600 for alkane production constructs every 24 hours for a 72 hour period, producing a growth curve.<br />
[[File:Washington growthcurve.png|center|450px|thumb|Growth curve showing that cells expressing FabH2 are growth deficient. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 ADC] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 AAR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 DrR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2]<br />
]]<br />
After 24 hours, FabH2 producing cells had barely grown at all, indicating a severe growth deficiency. The cells were able to rapidly grow after 24 hours, presumably due to a mutation that counteracted the negative effects of FabH2. This toxicity could be due to branched chain fatty acid production, or due to the activity of FabH2 on straight chain substrates affecting cellular metabolism. This severe growth deficiency implies that FabH2 is being expressed, and has activity that is detrimental to cell growth. Due to FabH2's toxicity, any time that we coexpressed fabH2 and the PetroBrick on a high copy number, strong promter expression vector, total alkane yield was decimated. In cells expressing the normal PetroBrick, we were able to consistently Reduction of this toxicity should allow us to determine the effects of FabH2 production on alkane biosynthesis.<br />
<br />
=Reducing the Toxicity of FabH2.=<br />
We hypothesized that if we could reduce the toxicity of FabH2, we could see the production of alternative alkane products. The original FabH2 system placed the FabH2 gene under the control of a strong constituitive promoter. We thought that if we could reduce FabH2 expression, we could decrease toxicity and be able to see the effects of FabH2 production on the alkanes produced by the PetroBrick. We cloned FabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number 3k3 IPTG inducible vector]. By reducing copy number appproximetly 10 fold, and by having the ability to control expression, we thought that we could lessen toxicity and observe even chain length alkane production. We called this inducible, low expression construct the FabBrick, a modular add-on to the standard PetroBrick.<br />
=FabBrick Induction Results in Even Chain Length Alkane Production=<br />
The FabBrick was co-transformed with the Petrobrick in XL1-Blue E. coli. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/Petrobrick cultures, and on cells expressing only the PetroBrick.<br />
<br />
There was no significant difference between the GC peaks in the PetroBrick extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When FabH2 is induced, we see a strong new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinately, and expands the PetroBrick system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels.<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
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[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]<br />
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==References==<br />
1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.<br />
<br />
2. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
3. Branched-chain fatty acid biosynthesis in Escherichia coli. Smirnova N, Reynolds KA. J Ind Microbiol Biotechnol. 2001 Oct;27(4):246-51.<br />
<br />
=Parts Submitted=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2] This part contains the FabH2 coding sequence, codon optimized for ''E. coli''<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2-ADC-AAR] This part expresses FabH2, as well as AAR and ADC, the two genes responsible for alkane production.</div>Hargemhttp://2011.igem.org/Team:Washington/Alkanes/Future/FabH2Team:Washington/Alkanes/Future/FabH22011-10-26T17:35:23Z<p>Hargem: /* FabBrick Induction Results in Even Chain Length Alkane Production */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
__NOTOC__<br />
<br />
<big><big><big>'''<center>Alternative Alkane Products</center>'''</big></big></big><br />
<br />
=Background=<br />
[[File:Washington_branched_CoA.png|right|450px|thumb|The branched CoA substrates used by FabH2]]<br />
The basic alkane production system is incapable of making branched chain alkanes, as ''E. coli'' normally only makes straight chain fatty acids. In addition, our basic system is only able to make odd chain length alkanes, as E. coli only normally make even chain length fatty acids. We thought that if we could introduce an enzyme into PetroBrick expressing E. coli that results in the production of branched chain or odd chain length Fatty acids, we could cause the PetroBrick to produce branched chain or even change length alkanes. One protein whose expression has been previously shown ([[#References | [1]]]) to cause ''E. coli'' to produce branched chain and odd chain length fatty acids is FabH2 from ''Bacillus subtilis''. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in even chain length linear fatty acids. However, FabH2 has been hypothesized to initiate fatty acid biosynthesis using Isobutyryl-CoA and Isovaleryl-CoA( products from Valine and Leucine degredation), resulting in 2-methyl branched fatty acid production, as well as using a 3-carbon propanoyl-CoA, resulting in odd chain length fatty acids. If we could use FabH2 and the Petrobrick to get ''E. coli'' to produce both alkanes and branched chain or odd chain length fatty acids, we should theoretically be able to produce 2-methyl branched alkanes or even chain length fatty acids.<br />
<br />
=Methods=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2 ] was constructed from oligos( refer to [https://2011.igem.org/Team:Washington/Protocols/gene_assembly protocol]). FabH2 was then amplified to add an [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 Elowitz standard RBS]. This RBS-FabH2 construct was cloned into the 3' end of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] to express [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2 as well as AAR and ADC]. In addition, we cloned fabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number IPTG inducible vector].<br />
<br />
=FabH2 is toxic to ''E. coli''=<br />
Throughout our experiments, we observed that cells expressing FabH2 grew significantly slower than any of our other alkane producing cells. In order to quantify this effect, we measured OD600 for alkane production constructs every 24 hours for a 72 hour period, producing a growth curve.<br />
[[File:Washington growthcurve.png|center|450px|thumb|Growth curve showing that cells expressing FabH2 are growth deficient. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 ADC] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 AAR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 DrR] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2]<br />
]]<br />
After 24 hours, FabH2 producing cells had barely grown at all, indicating a severe growth deficiency. The cells were able to rapidly grow after 24 hours, presumably due to a mutation that counteracted the negative effects of FabH2. This toxicity could be due to branched chain fatty acid production, or due to the activity of FabH2 on straight chain substrates affecting cellular metabolism. This severe growth deficiency implies that FabH2 is being expressed, and has activity that is detrimental to cell growth. Due to FabH2's toxicity, any time that we coexpressed fabH2 and the PetroBrick on a high copy number, strong promter expression vector, total alkane yield was decimated. In cells expressing the normal PetroBrick, we were able to consistently Reduction of this toxicity should allow us to determine the effects of FabH2 production on alkane biosynthesis.<br />
<br />
=Reducing the Toxicity of FabH2.=<br />
We hypothesized that if we could reduce the toxicity of FabH2, we could see the production of alternative alkane products. The original FabH2 system placed the FabH2 gene under the control of a strong constituitive promoter. We thought that if we could reduce FabH2 expression, we could decrease toxicity and be able to see the effects of FabH2 production on the alkanes produced by the PetroBrick. We cloned FabH2 into a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K314103 low copy number 3k3 IPTG inducible vector]. By reducing copy number appproximetly 10 fold, and by having the ability to control expression, we thought that we could lessen toxicity and observe even chain length alkane production. We called this inducible, low expression construct the FabBrick, a modular add-on to the standard PetroBrick.<br />
=FabBrick Induction Results in Even Chain Length Alkane Production=<br />
The FabBrick was co-transformed with the Petrobrick in XL1-Blue E. coli. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/Petrobrick cultures, and on cells expressing only the PetroBrick.<br />
<br />
There was no significant difference between the GC peaks in the PetroBrick extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When FabH2 is induced, we see a strong new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinately, and expands the PetroBrick system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels.<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
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<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]<br />
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<br />
\<br />
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<br />
<br />
==References==<br />
1.Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.<br />
<br />
2. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
<br />
3. Branched-chain fatty acid biosynthesis in Escherichia coli. Smirnova N, Reynolds KA. J Ind Microbiol Biotechnol. 2001 Oct;27(4):246-51.<br />
<br />
=Parts Submitted=<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590034 FabH2] This part contains the FabH2 coding sequence, codon optimized for ''E. coli''<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590030 FabH2-ADC-AAR] This part expresses FabH2, as well as AAR and ADC, the two genes responsible for alkane production.</div>Hargem