Team:Nevada/Project/Results

Results Summary

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

A standard curve was found, using casamino acids at a concentration of 0.20% for I^q cells, allowing us to detect successful passage of glucose from cyanobacteria to E. coli. It was determined that we could increase growth of our E. coli by 10% with as little as 500 µM glucose or up to 60% with 2.5 mM. Stress testing of the apparatus was done, ensuring that it is not leaking. Several methods were tried to prevent passage of E. coli into the cyanobacteria chamber, while traveling through the dialysis tubing. Eventually heatshrink was used to cinch the dialysis tubing to the glass support and we successfully prevented cross-contamination during a 48 hour run of the apparatus.

Conclusions

The Nevada iGEM team set out to create a self-sustaining system where cyanobacteria, a photosynthetic bacteria, would be capable of producing glucose to feed E. coli and save 30-40% on the cost of producing genetically engineered products in E. coli. A co-cultivation system was designed where transformed E. coli and cyanobacteria could grow in the same media and E. coli could produce 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.