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Team:St Andrews/Omega-3-synthesis
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<p>Chart 1. Chart 1. C18:3 refers to an 18 carbon backbone with 3 double bonds. C18:2 and C18:1 would have 2 double bonds and 1 respectively. The upper portion of this figure shows the peaks that would be expected for the various fatty acid methyl esters.The lower 4 panels show mass spec fingerprints that are indicative of our fatty acids of interest. | <p>Chart 1. Chart 1. C18:3 refers to an 18 carbon backbone with 3 double bonds. C18:2 and C18:1 would have 2 double bonds and 1 respectively. The upper portion of this figure shows the peaks that would be expected for the various fatty acid methyl esters.The lower 4 panels show mass spec fingerprints that are indicative of our fatty acids of interest. | ||
| - | <p>Chart 2. All of the cellular lipids were extracted and their fatty acid methyl esters produced. These were analysed by mass spec. In the control cells no C18:2 is present. When Delta 6 is expressed, a peak indicative of C18:2 is present. However, the primary fatty acid is still C18:1. With the expression of Delta 12 | + | <p>Chart 2. All of the cellular lipids were extracted and their fatty acid methyl esters produced. These were analysed by mass spec. In the control cells no C18:2 is present. When Delta 6 is expressed, a peak indicative of C18:2 is present. However, the primary fatty acid is still C18:1 - with the bouble bond present at position 9. With the expression of Delta 12 the C18:1(9) is processed and we get great abundance of C18:2. The lowest graph shows that Delta 12 expressed with Delta 15 acts in a complementary fashion and C18:3 - Omega 3 - is produced. </p> |
<p>Chart 3. The cellular membranes were extracted by ultracentrifugation and then their lipids were analysed by mass spec. As in chart 2 it is confirmed that expression of Delta 12 and Delta 15 lead to the production of omega 3 fatty acids.</p> | <p>Chart 3. The cellular membranes were extracted by ultracentrifugation and then their lipids were analysed by mass spec. As in chart 2 it is confirmed that expression of Delta 12 and Delta 15 lead to the production of omega 3 fatty acids.</p> | ||
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Revision as of 21:29, 24 September 2012
Omega-3 fatty acid synthesis
Introduction
ω-3 Fatty acids are an essential component of our diet and are paramount to maintaining human health. Our team is recreating this synthetic pathway in E. coli, using genes from the cyanobacteria Synechocystis and the trypanosomatid Leishmania major. Combining the DNA code for elongase and desaturase enzymes, we can convert the plain fatty acid of E. coli into highly valuable ω-3 fatty acids.
Project Description
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Synechocystis sp. -
Trypanosome cruzi -
Leishmania major
Omega-3 fatty acids are an essential part of the human diet (Bender, Bender, 1999). Human beings, as all larger organisms, cannot synthesize ω-3 fatty acids, This is due to a lack of the enzyme Δ15 desaturase, which creates a double bond at the 15th carbon of a long-chain fatty acid. Certain microrganisms, such as microalgae and cyanobacteria, do contain this desaturase and can thus directly synthesize ω-3 fatty acids (Arts et al, 2009). ω-3 fatty acids then enter the food chain – algae are eaten by fish, and seafood is sbsequently the main source of ω-3 for humans(Tonon et al, 2002).
However, the current economic policies of overfishing are a serious contributor to marine biodestruction. As the human population is estimated to rise to 9.1 billion by 2050 (Cohen, 2003), pressure on fish stock will increase. Additionally, global warming will reduce the availability of ω-3 (Arts et al, 2009): at higher temperatures, microalgae produce less ω-3 desaturated fatty acids. Desaturated carbon chains cause a lower melting temperature in the membrane, which the microorganism wants to avoid by synthesizing more saturated fatty acids in their membranes (Garwin, Cronan, 1980). Thus, the combination of declining fish stock and a decrease in overall ω-3 fatty acids is making the supply for human nutrition a relevant issue.
Harvesting algae directly is costly and ineffective (Borowitzka, 1997). There is much potential in expressing a metabolic pathway for ω-3 fatty acid synthesis in E. coli, which is cheaper and more accessible.
Synthesizing the pathway
E. coli naturally synthesize poly-unsaturated fatty acids up to a carbon chain length of 18, with a single desaturation at the 9th carbon (18:1) (Marr, Ingraham, 1969). Valuable ω-3 fatty acids require 3 double bonds, and need to have a chain length between 18 and 22 carbons. The double bonds needs to start at the thrid carbon, counting from the end of the carbon chain.
In order to have
The genes for Δ12, Δ15 (ω6) and Δ6 were obtained from Synechocystis sp., a cyanobacterium. The trypanosomatid Leishmania major provided the DNA for the ELO 6 gene. Additionally, we used Trypanosome cruzi as a secondary source of Δ12. (cf. Fig. 2)
However, our first successful ligations of Δ12 did not yield us with the expected 18:2 fatty acid. We hypothesized that E. coli’s inherent 18-carbon chain fatty acid might not be suited as a substrate for Δ12 – perhaps the double bond is in a different position. Therefore, we "fed" our cells with suitable 18:1, to then observe 18:2 fatty acid in the mass spec results!
The below figure shows the results of a PCR extraction of our genes of choice, done with GoTaq HotStart PCR kit at 2 different annealing temperatues: Δ12 (48°C) - Δ12 (56°C) - Δ15 (48°C) - Δ15 (56°C) - Δ6 (48°C) - Δ6 (56°C).
Methods
Sequences
Primers
All primers are notated 5' to 3'. Initially, we worked with NdeI and XhoI as the restriction sites.
Lipid analysis
Lipids were extracted from transformed E. coli using the Blight/Dyer method. (Please refer to the Lipid extraction lab book entry for further detail.)
We then analyzed the the fatty acid content of the transformed cells to determine whether it differed from the previously analyzed background lipid composition. This was done using mass spectrometry.
Mass Spectrometry
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Chart 1: "Fatty acid methyl ester analysis"
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Chart 3: "Lipid extraction analysis"
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Chart 4: "Membrane assay"
Chart 1. Chart 1. C18:3 refers to an 18 carbon backbone with 3 double bonds. C18:2 and C18:1 would have 2 double bonds and 1 respectively. The upper portion of this figure shows the peaks that would be expected for the various fatty acid methyl esters.The lower 4 panels show mass spec fingerprints that are indicative of our fatty acids of interest.
Chart 2. All of the cellular lipids were extracted and their fatty acid methyl esters produced. These were analysed by mass spec. In the control cells no C18:2 is present. When Delta 6 is expressed, a peak indicative of C18:2 is present. However, the primary fatty acid is still C18:1 - with the bouble bond present at position 9. With the expression of Delta 12 the C18:1(9) is processed and we get great abundance of C18:2. The lowest graph shows that Delta 12 expressed with Delta 15 acts in a complementary fashion and C18:3 - Omega 3 - is produced.
Chart 3. The cellular membranes were extracted by ultracentrifugation and then their lipids were analysed by mass spec. As in chart 2 it is confirmed that expression of Delta 12 and Delta 15 lead to the production of omega 3 fatty acids.
Biobricks
TODO
References
ARTS, et al, 2009. Lipids in Aquatic Ecosystems. New York. Springer.
BENDER D. A. and BENDER, A. E, 1999. Benders' dictionary of nutrition and food technology. Cambridge: CRC Press.
COHEN, J., 2003. Human Population: The Next Half Century. Science, New Series, Vol. 302, No. 5648. Pp. 1172-1175.
GARWIN J. L. and CRONAN J. E. Jr, 1980. Thermal modulation of fatty acid synthesis in Escherichia coli does not involve de novo enzyme synthesis. J Bacteriol, 141(3): 1457–1459.
LIVORE V., TRIPODI K., UTARRO A., 2007. Elongation of polyunsaturated fatty acids in trypanosomatids. FEBS Journal, 274: 264–274.
MARR A., INGRAHAM J., 1969. Effect of temperature on the composition of fatty acids in Escherichia coli. J. Bactiol. 84(6). Pp. 1260–1267.
TONON T., et al, 2002. Long chain polyunsaturated fatty acid production and partitioning to triacylglycerols in four microalgae. Phytochemistry, Vol 61 Iss 1. Pgs 15-24.
