Team:Washington/Parts

From 2011.igem.org

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<center><big><big><big><big>'''Data Page'''</big></big></big></big></center><br><br>
<center><big><big><big><big>'''Data Page'''</big></big></big></big></center><br><br>
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<center><gallery caption="An Overview of the 2011 UW iGEM Teams Summer Projects" widths="250px" heights="400px" perrow="3">
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<center><big>An overview of the 2011 UW iGEM team's summer projects</big></center>
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Image:Diesel Production for Wiki.png|'''Make It: Diesel Production'''<br>We achieved production of alkanes, the main component of diesel, in bacteria using our novel PetroBrick.
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[[File:Washington_Spacer.jpg|1px]]
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Image:Gluten Destruction for Wiki.png|'''Break It: Gluten Destruction'''<br>Gluten intolerance prevents We reengineered a protease, active at low pH, for strongly enhanced activity against PQLP, the most common motif in the immunogenic peptide.
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[[Image:UW Diesel Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]
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Image:Gibson Assembly and Magnetosomes for Wiki.png|'''The GibsonBricks ToolKit'''<br>We completed two iGEM toolkits. In the first we created five Gibson Cloning versions of traditional biobrick vectors, and in the second we cloned the genes essential for magnetosome formation and transformed two into ''E. coli''
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[[File:Washington_Spacer.jpg|20px]]
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[[Image:UW Gluten Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]
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</gallery></center>
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[[File:Washington_Spacer.jpg|20px]]
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[[Image:UW Toolkits Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Background]]
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[[File:Washington_Spacer.jpg|5px]]
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{| style="background: white; text-align: center; width: 970px;" align="center" border="0"
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| '''Make It:  Diesel Production'''
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| '''Break It:  Gluten Destruction'''
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| '''iGEM Toolkits: Gibson Assembly<br>and Magnetosomes'''
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|}
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{| style="background: white; text-align: left; width: 965px;" border="0"
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|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.
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|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.
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|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''.
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|}
='''Data Summary'''=
='''Data Summary'''=
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==Data for Favorite New Parts==
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==''Data for Favorite New Parts''==
==='''Diesel Production'''===
==='''Diesel Production'''===
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[http://partsregistry.org/Part:BBa_K590025 BBa_K590025 '''"The Petrobrick"''']
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:: '''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.
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:: '''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
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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 in psb1C3 regulated by a high constitutive promoter.
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590026 BBa_K590026 '''ADC-PSB1C3-High constitutive''']
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The aldehyde decarbonylase behind a high constitutive promoter and an Elowitz RBS. This was an important negative control when testing for alkane production.
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590027 BBa_K590027 '''AAR-PSB1C3-High consititutive''']
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The acyl-ACP reductase behind a high constitutive promoter and an Elowitz RBS. This was an important negative control when testing for alkane production.
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 BBa_K590031 '''Synechococcus elongatus PCC7942 Aldehyde Decarbonylase''']
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This part encodes an enzyme (Aldehyde Decarbonylase (ADC)) that removes the carbonyl group (C=O) from a fatty aldehyde, yielding an alkane one carbon shorter than the original aldehyde and a molecule of formate.  This part has been codon-optimized for ''E. coli''.
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 BBa_K590032 '''Acyl-ACP Reductase''']
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This part encodes an enzyme that reduces cellular fatty acyl-ACPs, from the bacterial fatty acid biosynthesis pathway, into fatty aldehydes. This part has been codon optimized for ''E. coli''.
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==='''Gluten Destruction'''===
==='''Gluten Destruction'''===
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590021 BBa_K590021: '''Kumamolisin-As''']
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:: '''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.
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An enzyme from the sedolisin family native to ''Alicyclobacillus sendaiensis'' with known collagenase activity at low pH and elevated temperatures.
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590022 BBa_K590022: '''Kumamolisin-As_G319S, D358G, D368H''']
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A mutated Kumamolisin-As enzyme aimed to combat gluten intolerance by increased activity with the PQLP peptide, an antigenic epitope in gliadin. This mutant has point mutations at residues 319, 358, and 368 from Glycine to Serine, Aspartate to Glycine, and Aspartate to Histidine, respectively.
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590023 BBa_K590023: '''Kumamolisin-As_N291D''']
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A mutated Kumamolisin-As enzyme aimed to combat gluten intolerance by increased activity with the PQLP peptide, an antigenic epitope in gliadin. This mutant has a point mutation at residue 291 from Asparagine to Aspartate.
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590024 BBa_K590024: '''Kumamolisin-As_S354N, D358G, D368H''']
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A mutated Kumamolisin-As enzyme aimed to combat gluten intolerance by increased activity with the PQLP peptide, an antigenic epitope in gliadin. This mutant has point mutations at residue 354, 358, and 368 from Serine to Asparagine, Aspartate to Glycine, and Aspartate to Histidine, respectively.
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==='''Gibson Assembly Toolkit'''===
==='''Gibson Assembly Toolkit'''===
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590010 BBa_K590010 '''pGA1A3_pLacGFP''']
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::'''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.
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This is a high copy plasmid backbone which has ampicillin resistance, with pLac promoter and GFP.
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590011 BBa_K590011 '''pGA1C3_pLacGFP''']
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This is a high copy plasmid backbone which has chloramphenicol resistance, with pLac promoter and GFP.
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590012 BBa_K590012 '''pGA4C5_pLacGFP''']
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This is a low copy plasmid backbone which has chloramphenicol resistance, with pLac promoter and GFP.
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590013 BBa_K590013 '''pGA4A5_pLacGFP''']
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This is a low copy plasmid backbone which has ampicillin resistance, with pLac promoter and GFP .
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590014 BBa_K590014 '''pGA3K3_pLacGFP''']
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This is a medium copy plasmid backbone which has kanamycin resistance, with pLac promoter and GFP.  
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==='''Magnetosome Toolkit'''===
==='''Magnetosome Toolkit'''===
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590015 BBa_K590015 '''sfGFP_mamK_pGA1C3''']
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:: '''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.
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This part consists of mamK gene from Magnetospirillum magneticum strain AMB-1, sfGFP in the backbone of pGA1C3. mamK was previously reported for its importance proper magnetosome chain organization in magnetic bacteria; it is bacterial actin-like cytoskeleton protein required for proper alignment of the magnetosomes in a chain.
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590016 BBa_K590016 '''sfGFP_mamI_pGA1C3''']
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This part consists of mamI gene from Magnetospirillum magneticum strain AMB-1, sfGFP in the backbone of pGA1C3. MamI is a membrane-localized protein that localizes magnetic vesicles to the surface of cells, thus forming characteristic magnetosome chains.
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==Data for Existing Parts==
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:: '''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.
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Fill Me In
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# [http://partsregistry.org/Part:BBa_J45119:Experience Experience] - '''Wintergreen odor enzyme generator, BBa_J45119''' (MIT, iGEM 2006): 98 out of 100 volunteer subjects standing up to 5 feet away from the bacterial cultures could distinguish wintergreen-producing bacteria from negative controls.
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# [http://partsregistry.org/Part:BBa_J61110:Experience Experience] - '''RBS, BBa_J61110''' (Arkin Lab, 2007): Of the 5 RBS Parts we tested, this RBS works best for expressing yellow fluorescent protein-tagged BAR
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==''Data for Existing Parts''==
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==Improved Parts==
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::'''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.
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Fill Me In
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::'''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.
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# [http://partsregistry.org/Part:BBa_XXXXX Main Page] - '''Air Freshilizor, BBa_XXXXX''': Our mathematical model predicts that the threshold of activation is 10 parts per billion, the concentration of Butanethiol that humans can typically smell
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==''Improved Parts''==
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::'''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.
='''All Submitted Parts'''=
='''All Submitted Parts'''=
<center><groupparts>iGEM011 Washington</groupparts></center>
<center><groupparts>iGEM011 Washington</groupparts></center>

Latest revision as of 18:09, 26 October 2011


Data Page


An overview of the 2011 UW iGEM team's summer projects

Washington Spacer.jpg UW Diesel Front Page.png Washington Spacer.jpg UW Gluten Front Page.png Washington Spacer.jpg UW Toolkits Front Page.png Washington Spacer.jpg

Make It: Diesel Production Break It: Gluten Destruction iGEM Toolkits: Gibson Assembly
and Magnetosomes
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. 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. 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.


Data Summary

Data for Favorite New Parts

Diesel Production

1, 2. BBa_K590025: The PetroBrick - A modular and open platform for the biological production of diesel fuel. The PetroBrick consists of AAR and ADC, each behind a standard Elowitz RBS. All of this is under regulation by a high constitutive promoter in pSB1C3.
3 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 low copy number IPTC inducible vector These are converted into even chain length alkanes by BBa_K590025 the PetroBrick, completing the spectrum of linear alkane compounds that can be produced using the

Gluten Destruction

4. 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.

Gibson Assembly Toolkit

5. BBa_K590010: pGA1A3, BBa_K590011: pGA1C3, BBa_K590012: pGA4C5, BBa_K590013: pGA4A5, 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.

Magnetosome Toolkit

6. 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.
7. 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.

Data for Existing Parts

8. 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.
9. 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.

Improved Parts

10. BBa_K590059, BBa_K590060, BBa_K590061: LuxC, D, and E (Cambridge, iGEM 2010) - Formerly part of the 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.

All Submitted Parts

<groupparts>iGEM011 Washington</groupparts>
Retrieved from "http://2011.igem.org/Team:Washington/Parts"