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 acyltrans- ferase (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. 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 growing in our lab. E. Coli was the ideal bacterium to use in the co-culture as it has an incredible amount of research supporting it and 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.

C. minutissima 

     The C. minutissima was to be genetically modified with enzymes involved in lipid production, which included a glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltrans- ferase (LPAAT), and diglyceride acyltransferase (DGAT). These gene constructs were also designed in  SnapGene and synthesized by  IDT. The constructs were ligated onto a DinoIII plasmid from our previous iGEM project and induced using the CAMV promoter to allow for expression in algae.      

Test

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.

Plasmid Transformations 

     We picked a colony from each plate and the culture out overnight in our shaker to perform midi preps the next day. We also did a colony PCR using colonies from the plate in order to verify the presence of our ligated product. The readings on the midi prep and the gel from the colony PCR were good so we were certain that we should proceed to perform a protein extraction and western blot to detect any protein expression.

Proposed Implementation

     After we have successfully proven that co-culture between C. minutissima and E. coli leads to increased amounts of lipids in the microalgae, we plan to market the project idea to the National Renewable Energy Laboratory (NREL). We have discovered through our human practice interaction with them that they are interested in the co-culture methods to increase lipid production, so
our project would hopefully be something they look forward to taking on.

Sources 

     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.