Team:Nevada/Project/Results

Results Summary

The Nevada iGEM team set out to create a self-sustaining system where cyanobacteria, a photosynthetic bacteria, is capable of producing glucose that will feed E. coli and save 30-40% on the cost of production. A co-cultivation system was to be designed where transformed E. coli and cyanobacteria could grow in the same media and E. coli could product genetically engineered products, such as biofuels. Although this was a very ambitious project, we accomplished many of our goals and provided an excellent foundation to build on for next years iGEM competition.

E. coli

One of the primary goals of the E.coli group was to transform E. coli with genes that express biofuels, including medium chain free fatty acids and ethanol. Medium chain fatty acids were produced in E. coli at a level 2.5 fold higher than background by expressing the Bay Laurel thioesterase gene. Ethanol was also produced using a generator containing both the pyruvate decarboxylase and alcohol dehydrogenase genes. A total of 0.02% ethanol was produced.

An additional goal for the E. coli team was to express the above genes under an inducible promoter, trc, that is insensitive to changes in glucose levels. Unfortunately, we were not successful in accomplishing this goal.

Cyanobacteria

Each gene part of the two knockout/operon insertion constructs has been successfully isolated and identified by gel electrophoresis. Additionally, the invertase (inv) and glucose-facilitator transport genes (GLF) were confirmed by sequence. The success rate of assembling gene parts by Gibson assembly is very low for two parts alone, let alone four. For this reason we have begun experiments using PCR to build the internal constructs of each KO/operon prior to attempting Gibson and other variations of "chewback" DNA technology. Furthermore, exploration of which enzymes are particularly well-suited for our unique constructs should lead to increased success rates.

Co-cultivation