We intend to put the GABA producing proteins in E. coli Nissle 1917, creating a probiotic GABA supplement that functions in human intestine, with milk as the switch to open or close the production. In this way, the production of GABA will be simplified since E. coli Nissle is edible.
We chose the pTac promoter, a hybrid of Lac and Trp promoters, induce GABA production in E. coli Nissle in the human intestine in the presence of lactose from consumed milk. The promoter can be induced by both lactose and IPTG, where LacI represses transcription of up stream genes in this part, which are gadR, gadC, and gadA. The ribosomal binding site (RBS) for gadRCA with a relative strength of 300, 300, 3900 respectively are calculated by RBS Calculator from Salis Lab.
Fig. 2 Second Approach: BBa_K2326004
Because E. coli Nissle has not been used as an engineered strain until recent years, it cannot express large amount of foreign genes from a plasmid and failed to execute all the designs we had on our first attempt. So we cut our original design, leaving the most important enzyme, gadA, alone, which catalyzed GABA transforming from glutamate. We also changed the RBS of gadA into B0034, the most consensus sequence for E. coli. Moreover, we added GFP at the end of the circuit to simplify the process of assaying protein production.
Fig. 3 Third Approach: BBa_K2326005 and BBa_K2326006
The second device we designed enabled us to see an estimated amount of gadA expressed, but the negative result from the Amino Acid Analyzer (see demonstrate) suggest the GABA produced in our project may flow back to the TCA cycle. As a result, we designed the third and fourth device as shown above. For BBa_K2326005, we added a His tag after gadA so we can extract the enzyme out and prevent it from being subjected to endogenous processes, and then produce GABA from the isolated protein. BBa_K2326006 enabled us to quantify the expression rate of gadA since the protein is synthesized with GFP.