Although bacteria can naturally synthesize GABA, we wanted to increase expression of the gadB gene and subsequently GABA production in order to give our intended probiotic, Lactobacillus plantarum, a more potent medicinal quality, with the idea that this GABA-overproducing probiotic can then be consumed by patients with bowel disorders, hypertension or anxiety (1). Overexpression of the gadB gene will be accomplished by placing it under the control of either the P8 or P32 constitutive promoters from Lactococcus lactis (2).

To make our GABA-producing probiotic, we ultimately needed to assemble a GABA overexpression cassette plasmid. The intention is that bacteria containing this GABA overexpression cassette plasmid should produce high levels of GABA. In order to assemble this plasmid, we decided to utilize the Golden Gate Assembly method. In short, Golden Gate Assembly is a relatively new cloning method that allows for the creation of a multi-part DNA assembly (i.e. cassette plasmid) in a single reaction through the use of DNA parts containing specific, predefined suffixes and prefixes with recognition sites for Type IIs restriction enzymes (e.g. BsmBI and BsaI). The specificity of these suffixes and prefixes provides directionality of the desired DNA parts during the assembly process. For our purposes, we used the MoClo Yeast Tool Kit developed by John Dueber (3).

We decided to first assemble and test our Golden Gate plasmids in E. coli, which was chosen due to the ease in which we could genetically manipulate it. We then wanted to use these Golden Gate plasmids to genetically manipulate L. plantarum. This part of the project required us to assemble a Golden Gate compatible shuttle vector (that is replicable in both E. coli and L. plantarum ) and transform L. plantarum. Our experimental results are detailed below.