Team:MSU-Michigan/Design

Michigan State University iGEM 2016

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Design

Research is ongoing to find ways to use cyanobacteria in commercial production of carbon compounds such as sugar, biofuels, and pharmaceutical ingredients. Since cyanobacteria obtain their energy through photosynthesis, producing these compounds in industrial quantities may require outdoor production. We can also use cyanobacteria in this experiment as a predictor of how certain plant cells will react in lower temperatures, helping to predict how crops will be affected by certain conditions.¹ Our project was designed to look at ways to improve cyanobacteria’s chilling tolerance which opens the possibility of utilizing colder and less desirable geographical regions for production.

In our research, we found that “the chilling tolerance of cyanobacteria can be enhanced by genetic manipulation of fatty acid desaturation”.² A plant-type desaturase (desA) affects the lipids in the membrane through the addition of double bonds in the fatty acids. What if we were able to insert that protection into a cyanobacteria that is widely-used for the production of green commodities? Our project was designed to do just that.

• Design and construct multiplexed chemostat to allow cyanobacterias to grow continuously, as they would in an industrial setting.
• Express desA protein from Synechocystis sp. PCC6803, a species of cyanobacterium that is able to survive in a large range of conditions, in Synechococcus elongatus PCC7942, another species of cyanobacterium that is widely used and studied for the production of the green commodities listed above.
• Clone desA plasmids.
• Transform plasmid into Synechococcus elongatus PCC7942 to create engineered cyanobacteria.
• Using Gibson Assembly, insert cloned desA genes into Synechococcus elongatus PCC 7942. Gibson Assembly usage provided a tightly-controlled riboswitch vector.
• Place engineered cyanobacteria and wild types of cyanobacteria into the sterile chemostat culture chambers. Place chemostat in various temperatures for continuous growth.
• Determine viability of engineered and wild strains of cyanobacteria after being subjected to various temperatures.
• Examine lipid membrane for differences in fatty acid make-up.

Future work on this project will focus on gradually decreasing the temperatures to further increase the freeze tolerance of the cyanobacteria. We also would like to use immunofluorescence to determine whether the desA is cytosolic- or membrane-bound, and use high-resolution microscopy methods to see changes in the outer chloroplast membrane.

For more information on the multiplexed chemostat, please refer to our Hardware page.

¹https://www.ncbi.nlm.nih.gov/pubmed/2118597
²Ibid.