Team:Korea U Seoul/Project

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Overall project

The goal of our project is to produce alkane chains from glucose molecules. In nature, numerous biochemical pathways and enzymes exist, making life adoptable to even extreme conditions such as volcanic regions. We focused on biochemical pathways, enzymes of glucose metabolism and luminescene luciferase from Vibrio harveyi to achieve our goal. Based on glycolysis, pyruvate oxidation, enzymes coded in luciferase genes (lux operon) and FAD from cyanobacteria, glucose is turned into alkane chain of about 14 carbon atoms in length. Synthesized fuel is functionally identical to natural petroleum and can be used as bioenergy. Produced alkane chain is part of a carbon circulation cycle as it is synthesized from glucose, in vivo. The fuel is relatively environment-friendly, unlike ordinary petroleum which increases CO2 concentration in the atmosphere. Though the production of alkanes using bioblock could be not satisfied commercially, succeeding in the synthesis of alkane chains from glucose nevertheless will show another method of producing alternative energy source. Therefore, the success of this research will contribute to global effort in reducing atmospheric CO2 levels.

Project Abstract

Synthesis of Synthetic Micro-Alkanes (“Synfuels”) in Engineered Escherichia coli
Our team concentrated on finding the solution to the world’s diminishing natural oil and gas resources and greenhouse gas emissions. The aim of our project is the production of biofuels, alkanes, using bacterial cells as factories. Alkanes, so called “Green” hydrocarbon fuels, are chemically energetically the same as petroleum-based fuels, thus no penalty for use of conventional engines is encountered from their use. For alkane biosynthesis, we designed a synthetic circuit using bacterial bioluminescence system and aldehyde decarbonylase from Vibrio harveyi and cyanobacteria, respectively. Free fatty acids in the cells firstly are reduced and converted to fatty aldehydes by Lux C, Lux D and Lux E and then fatty aldehydes finally are decarbonylated and turned into alkanes.

Project Details

E.coli K27 strains

- The purpose of our team is to synthesize alkanes from microorganisms. E.coli K27 is a suitable host for the production of alkanes because it is a FadD mutant(△fadD).

- In our step of alkanes synthesis, the fatty acids are important intermediates. Commonly, E.coli cells contain a single acyl-CoA synthetase, which activates the conversion of free fatty acid to acyl-CoA thioester. However, E.coli K27, a FadD mutant, lacks acyl-CoA synthetase activity, which prevents substrate or product degradation by the host. So, E.coli K27 accumulates fatty acids inside the cell, and finally we can get more alkanes than other E.coli strains.

Two-carbon compounds and fatty acids as carbon sources

Synthetic pathway

Lux genes

- The genes from bioluminescence operons have been identified, and we use some structural genes (luxC, D, and E genes). They code for the polypeptides of the fatty acid reductase system responsible for synthesis of the fatty aldehyde substrate.

- There are 5 lux genes that we used. lux A, B, C, D, and E were used. lux A and lux B codes Luciferase alpha and beta subunit respectively. lux C codes Reductase, lux D codes Acyl-transferase, and lux E codes Synthetase. Luciferase alpha and beta subunit function is catalysis of the bioluminescence reaction(FMNH2 + O2 + aldehyde -> light). Reductase's function is NADPH-dependent reduction of activated fatty acyl groups to aldehyde. Acyl-transferase's function is generation of fatty acids(tetradecanoic acid) for the luminescence system. Lastly, Synthetase's function is ATP-dependent activation of fatty acids.

Bioluminescence

Manipulation of Lux genes

Detection of hydrocarbon

We are currently having difficulty in detecting the final product, C14.

- Detection of C14 by TLC is difficult. It is not yet providing good enough results. Apparently, this is due to the fact that saturated hydrocarbons don't react with any other compounds.

- By incorporating BBa_K32599 in luxCDEG, we may be able to detect hydrocarbon.

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-|You can write a background of your team here.  Give us a background of your team, the members, etc.  Or tell us more about something of your choosing.
 |[[Image:Korea_U_Seoul_logo.png|200px|right|frame]]
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-''Tell us more about your project.  Give us background.  Use this is the abstract of your project.  Be descriptive but concise (1-2 paragraphs)''
 |[[Image:Korea_U_Seoul_team.png|right|frame|Your team picture]]
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 == '''Overall project''' ==
-<p> The goal of our project is to produce alkane chains from glucose molecules. In nature, numerous biochemical pathways and enzymes exist, making life adoptable to even extreme conditions such as volcanic regions. We focused on biochemical pathways, enzymes of glucose metabolism and luminescene luciferase from ''Vibrio harveyi'' to achieve our goal. Based on glycolysis, pyruvate oxidation, enzymes coded in luciferase genes (lux operon) and FAC from cyanobacteria, glucose is turned into alkane chain of about 14 carbon atoms in length. Synthesized fuel is functionally identical to natural petroleum and can be used as bioenergy. Produced alkane chain is part of a carbon circulation cycle as it is synthesized from glucose, in vivo. The fuel is relatively environment-friendly, unlike ordinary petroleum which increases CO2 concentration in the atmosphere. Though the production of alkanes using bioblock could be not satisfied commercially, succeeding in the synthesis of alkane chains from glucose nevertheless will show another method of producing alternative energy source. Therefore, the success of this research will contribute to global effort in reducing atmospheric CO2 levels. </p>
+<p> The goal of our project is to produce alkane chains from glucose molecules. In nature, numerous biochemical pathways and enzymes exist, making life adoptable to even extreme conditions such as volcanic regions. We focused on biochemical pathways, enzymes of glucose metabolism and luminescene luciferase from ''Vibrio harveyi'' to achieve our goal. Based on glycolysis, pyruvate oxidation, enzymes coded in luciferase genes (lux operon) and FAD from cyanobacteria, glucose is turned into alkane chain of about 14 carbon atoms in length. Synthesized fuel is functionally identical to natural petroleum and can be used as bioenergy. Produced alkane chain is part of a carbon circulation cycle as it is synthesized from glucose, in vivo. The fuel is relatively environment-friendly, unlike ordinary petroleum which increases CO2 concentration in the atmosphere. Though the production of alkanes using bioblock could be not satisfied commercially, succeeding in the synthesis of alkane chains from glucose nevertheless will show another method of producing alternative energy source. Therefore, the success of this research will contribute to global effort in reducing atmospheric CO2 levels. </p>
@@ Line 35: / Line 37: @@
 '''Synthesis of Synthetic Micro-Alkanes (“Synfuels”) in Engineered ''Escherichia coli'''''
 <br>
-Our team concentrated on finding the solution to the world’s diminishing natural oil and gas resources and greenhouse gas emissions. The aim of our project is the production of biofuels, alkanes, using bacterial cells as factories. Alkanes, so called “Green” hydrocarbon fuels, are chemically energetically the same as petroleum-based fuels, thus no penalty for use of conventional engines is encountered from their use. For alkane biosynthesis, we designed a synthetic circuit using bacterial bioluminescence system and aldehyde decarbonylase from Vibrio harveyi and cyanobacteria, respectively. Free fatty acids in the cells firstly are reduced and converted to fatty aldehydes by LuxC, LuxD and LuxE and then fatty aldehydes finally are decarbonylated and turned into alkanes.
+Our team concentrated on finding the solution to the world’s diminishing natural oil and gas resources and greenhouse gas emissions. The aim of our project is the production of biofuels, alkanes, using bacterial cells as factories. Alkanes, so called “Green” hydrocarbon fuels, are chemically energetically the same as petroleum-based fuels, thus no penalty for use of conventional engines is encountered from their use. For alkane biosynthesis, we designed a synthetic circuit using bacterial bioluminescence system and aldehyde decarbonylase from ''Vibrio harveyi'' and cyanobacteria, respectively. Free fatty acids in the cells firstly are reduced and converted to fatty aldehydes by ''Lux C'', ''Lux D'' and ''Lux E'' and then fatty aldehydes finally are decarbonylated and turned into alkanes.
 == '''Project Details''' ==
+*''E.coli'' K27 strains
+:- The purpose of our team is to synthesize alkanes from microorganisms. ''E.coli'' K27 is a suitable host for the production of alkanes because it is a FadD mutant(△''fadD'').
+:- In our step of alkanes synthesis, the fatty acids are important intermediates. Commonly, ''E.coli'' cells contain a single acyl-CoA synthetase, which activates the conversion of free fatty acid to acyl-CoA thioester. However, ''E.coli'' K27, a FadD mutant, lacks acyl-CoA synthetase activity, which prevents substrate or product degradation by the host. So, ''E.coli'' K27 accumulates fatty acids inside the cell, and finally we can get more alkanes than other ''E.coli'' strains.
-We believed that there exists difficulty in producing Wax ester. Therefore, we read carefully several dissertations in order to find a compound that we can use as biofuel. One of our members came up with an idea after finding a picture(shown below) about a synthetic pathway in fluorescing bacteria.
+*Two-carbon compounds and fatty acids as carbon sources
+[[File:E.coli picture.jpg|thumb|Synthetic pathway|500px|center]]
-[[File:fluorescing bacteria.jpg|frame less|400px|center]]
+*Lux genes
+:- The genes from bioluminescence operons have been identified, and we use some structural genes (''luxC'', ''D'', and ''E'' genes). They code for the polypeptides of the fatty acid reductase system responsible for synthesis of the fatty aldehyde substrate.
+:- There are 5 lux genes that we used. ''lux A'', ''B'', ''C'', ''D'', and ''E'' were used. ''lux A'' and ''lux B'' codes Luciferase alpha and beta subunit respectively. ''lux C'' codes Reductase, ''lux D'' codes Acyl-transferase, and ''lux E'' codes Synthetase. Luciferase alpha and beta subunit function is catalysis of the bioluminescence reaction(FMNH2 + O2 + aldehyde -> light). Reductase's function is NADPH-dependent reduction of activated fatty acyl groups to aldehyde. Acyl-transferase's function is generation of fatty acids(tetradecanoic acid) for the luminescence system. Lastly, Synthetase's function is ATP-dependent activation of fatty acids.
+[[File:lux.jpg|thumb|Bioluminescence|500px|center]]
-According to the picture above, it is possible to turn Acyl-ACP into a C14 aldehyde. We can then deduce from such a result the possibility of producing a C14 alkane from glucose, utilizing microorganisms as a synthetic machinery.
+[[File:arrows.jpg|thumb|Manipulation of Lux genes|500px|center]]
-By considering such possibilities, team members began searching for pathways that utilize two substances, which are:
+===Detection of hydrocarbon===
-. luxAB removed luxCDE from fluorescing bacteria
-. cyanobacteria's aldehyde decarbonylase gene
-Picture below depicts biofuel synthetic pathway.
+*We are currently having difficulty in detecting the final product, C14.
+:- Detection of C14 by TLC is difficult. It is not yet providing good enough results. Apparently, this is due to the fact that saturated hydrocarbons don't react with any other compounds.
+:- By incorporating BBa_K32599 in luxCDEG, we may be able to detect hydrocarbon.
-[[File:pic1.jpg|frame less|350px|center]]
+[[File:TLC method.jpg|frame less|500px|center]]
+[[File:TLC method2.jpg|frame less|500px|center]]
-This is what we came up with. It is a pathway that utilizes luminescence system and fatty acid reduction pathway already present within ''E.coli''. However, if a beta oxidation pathway is activated through acyl-CoA ligase, we will not be able to use carboxylic acid in the desired way. Therefore, we must use fadD to block beta oxidation.
-Based on these results, we carefully set up our research plan.
-[[File:iGEM plans.jpg|frame less||350px|center]]
-According to the plan above, we performed pre-research until July 22nd. By pre-research, we mean studying about luminescence system and lux operon activation in E.coli by reading dissertations, and also studying about biosynthetic pathways of alkanes. We also searched for the potential uses of C14. The potential uses we found are uses as energy source for various transportation. Fuel used today is usually extracted from the bottom of the oceans or miles below earth's surface. While natural petroleum increases atmospheric CO2 levels causing environmental problems, biofuel is synthesized by incorporating carbon from CO2, making it more environmentally friendly. Lastly, we searched for methods that can be used detect fatty acid, fatty aldehyde, and hydrocarbon, which are all intermediates in the pathway. Unfortunately, we were not able to find a method that is efficient.
-In order to prepare a proper medium, we searched for necessary nutrients in KACC(http://www.genebank.go.kr/) and KCTC(http://brc.re.kr/main.aspx). We then ordered and cultured ''Vibrio harveyi'' and cyanobacteria, followed by DNA extraction.
-After DNA extraction, we amplified target genes with PCR and as shown by the picture below, we made a circuit. Since luxCDE is a system not present in ''E.coli'', we must clone, insert it into ''E.coli'''s gene.
-After initial experiment, luminescence did not occur. We are re-examining the processes.
-[[File:pathway.jpg|frame less|550px|center]]
-==== Synthetic Fatty Acids Reduction Pathway ====
-=== Cloning of target genes ===
 == Results ==
-*''Vibrio harveyi'' KACC 14795 was cultivated in Marine broth 2216 at 26ºC.
-*''Vibrio harveyi'' genomic DNA extraction gave the following results.
-[[File:results1.jpg|frame less|550px|center]]
-*Cloning of ''luxCDABE'' to T7 promoter system by Ligation Independent Cloning gave the following results.
-[[File:results2.jpg|frame less|550px|center]]
-[[File:results3.jpg|frame less|550px|center]]
-*Expression tests were done on ''luxCDABE''.
-::- Induction : OD600(~0.8), 0.5mM IPTG, 37ºC, LB medium
-::- LuxC (54.8kDa), LuxD (34.2kDa), LuxA (40kDa), LuxB (36.3kDa), LuxE (42.9kDa)