Abstract
Greenhouse gas emissions arising from the combustion of fossil fuels are major contributors to climate change. Biofuels offer a carbon-neutral alternative: microalgae can be used to generate biofuels by absorbing carbon dioxide for photosynthesis. Our goal is to create a synthetic biology system that will increase lipid production from microalgae to serve as an intermediate in chemical reactions needed to create biofuels. Specifically, we will create a co-culture between C. minutissima microalgae and E. coli bacteria. To increase C. minutissima’s growth rate, E. coli will be genetically modified to produce indole-3-acetic acid (IAA). C. minutissima will be genetically modified to express glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), and diglyceride acyltransferase (DGAT) to increase lipid production. We hypothesize that this modified co-culture will result in a dense algae population with high lipid content, providing ideal parameters for biofuel production..
Engineering
Through the project, Synknlorum, we aimed to produce a co-culture product between the organisms E. Coli and C. Minutissima that were synthetically modified to maximize lipid production. The lipids produced by the algae could be used to produce biofuels on an industrial scale.
Design
For several decades fossil fuels have led the market as the main supply of energy for lighting, heating, and transportation. [1] As a non-renewable resource, the increasing population and need for more resources put fossil fuels at risk of scarcity. Burning these fossil fuels also contributes to 85% of Carbon dioxide released into the atmosphere. Carbon dioxide scatters infrared radiation, inducing the greenhouse effect and accelerating global warming and climate change. More sustainable and renewable alternatives to fossil fuel production have to be considered. Algal biofuel technology offers a more sustainable alternative. A lipid-producing microalgae, Chlorella minutissima has shown promise in increased neutral lipid production and biodiesel quality when co-cultured with the bacteria E. Coli. Therefore, we wanted to recreate this co-culture in our lab after synthetically modifying the C. Minutissima with genes for lipid-producing enzymes, LPAAT, DGAT, GPAT, and the E. Coli with a plant growth hormone, IAA. The two organisms would be grown together in a co-culture. We hypothesized that the algae in the co-culture would have an increased optimal density and lipid production, compared to an algae without the co-culture. The finalized product would be sent to the NERL to produce biofuels using their more advanced technology.
Build
We chose the microalgae, Chlorella minutissima due to its ability to grow in varied conditions, making it ideal for growth in our lab. E. Coli was the ideal choice as the bacterium to use in the co-culture as it has an incredible amount of research supporting it, and it has been used in similar co-cultures.
E. Coli
The engineered E. Coli was modified using two genes in the IAM pathway, tryptophan 2-monooxygenase (iaaM) and amidase 1 (ami1) to produce the plant growth hormone Indole-3-acetic acid (IAA). The genes were all designed using the SnapGene software and synthesized via the IDT sponsorship that iGEM provided us.
Test
Due to some complications in the purchase of the algae, we could not complete any algae transformations. We were able to produce our modified E. Coli constructs.
Plasmid Transformations
The ami1 and iaam coding sequences in the IDT plasmids were first individually transformed and grown on LB with ampicillin plates. The colonies were then picked and grown overnight, and midi preps were performed on the cultures to extract their DNA. Restriction digest using EcoRI and Xba1 isolated the coding sequences, which were then ligated into a PSB1C3 plasmid with promoters, RBS, and terminators chosen from the iGem 2022 DNA kit plate. The pSB1C3 plasmid, now ligated with the ami1 (BBa_K4493012) and iaam (BBa_K4493013) genes, was then used to transform E. coli which were then plated on LB with chloramphenicol.
Verification of constructs
A colony PCR was performed on the colonies that we grew overnight, and the results of the gel can be read below. The genes were also sent for sequencing to verify that they contained our genes of interest.
Western Blotting
We performed protein extraction and isolation on our transformed cultures. However, we chose not to use our samples for the western blotting as our technique while handling the protein isolation was poor. In the future, we aim to perfect our technique and proceed to perform a western blot on our two ligated plasmids.
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Abstract
1.Hannon M, Gimpel J, Tran M, Rasala B, Mayfield S. Biofuels from algae: challenges and potential. Biofuels. 2010 Sep;1(5):763-784. doi: 10.4155/bfs.10.44. PMID: 21833344; PMCID: PMC3152439.
2.Higgins, B. T., & VanderGheynst, J. S. (2014). Effects of Escherichia coli on mixotrophic growth of Chlorella minutissima and production of biofuel precursors. PloS one, 9(5), e96807. https://doi.org/10.1371/journal.pone.0096807
3.Varela Villarreal J, Burgués C, Rösch C. Acceptability of genetically engineered algae biofuels in Europe: opinions of experts and stakeholders. Biotechnol Biofuels. 2020 May 22;13:92. doi: 10.1186/s13068-020-01730-y. PMID: 32489422; PMCID: PMC7245023.