Team:Austin UTexas/Experiments



Experiments

Although bacteria can naturally synthesize GABA, we wanted to increase expression of the gadB gene and subsequently GABA production in order to imbue a more potent medicinal quality to our probiotic, with the idea that this GABA-overproducing probiotic can then be consumed by patients with bowel disorders or anxiety. To make our GABA-producing probiotic we first needed to assemble a GABA overexpression cassette plasmid using the Golden Gate assembly method. The intention here is that bacteria containing this GABA overexpression cassette plasmid should produce high levels of GABA. In short, Golden Gate Assembly is a 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 by John Dueber (Lee et al., 2015). The first part of our Golden Gate assembly workflow was part assembly, in which the gadB gene and the P8/P32 promoters were individually cloned into the entry vector pYTK001 (Fig. 1). The gadB gene and P8/P32 promoter sequences contain flanking BsmBI sites that produce overhangs compatible with those cut by BsmBI in the entry vector pYTK001. Thus, BsmBI cloning should result in part plasmids containing the gadB gene and P8/P32 promoters set within the pYTK001 backbone.

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Additionally, the BsmBI sites and overhangs in pYTK001 are flanking a gfp reporter gene. During the part assembly process, our DNA sequences of interest should replace this gfp reporter gene. This provides a phenotypic screen that allows us to visually see which transformant colonies are negative and potentially positive. Under UV illumination, positive colonies containing our intended part plasmid assembly did not exhibit fluorescence under the UV illumination, while negative colonies did (Fig. 2.). The non-fluorescent colonies on the part plasmid transformation plates were miniprepped and subsequently sequence verified.

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