Difference between revisions of "Team:Austin UTexas/Results"

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<p>Although bacteria can naturally synthesize GABA, we wanted to <b>increase expression of the <i>gadB</i> gene and subsequently GABA production in order to give our intended probiotic, <i>Lactobacillus plantarum</i>, a more potent medicinal quality</b>, with the idea that this GABA-overproducing probiotic can then be consumed by patients with bowel disorders, hypertension or anxiety (1). Overexpression of the <i>gadB</i> gene will be accomplished by placing it under the control of either the P8 or P32 constitutive promoters from <i>Lactococcus lactis</i> (2).
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<p>There are millions of species of bacteria1, few of which are well characterized. However, these non-model organisms perform cellular processes and are native to harsh biomes, and this makes them of great interest to researchers2. Synthetic biologists take great interest in engineering such bacteria, in an attempt to harness their natural abilities3. Because finding the best way to genetically modify an organism can be a laborious and resource-demanding process, we have developed a kit of modular plasmids, designed to quickly test multiple broad host range origins of replication (ORIs). To do this, we used a cloning technique called Golden Gate Assembly to build plasmids. Golden Gate Assembly uses Type IIs restriction enzymes, which allows researchers to pick the overhang each cut produces. Therefore, many parts can be assembled together at once, in a specific order. The plasmids are minimal and standardized4. They include barcode sequences and reporter genes to indicate which plasmid is which. A mixture of all the plasmids is transformed into a single sample of host bacteria, and the colored reporters allow the researcher to quickly determine which ORIs allow the host to replicate the plasmid.</p>
 
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<p>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).</p>
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<p>We decided to first assemble and test our Golden Gate plasmids in <i>E. coli</i>, 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 <i>L. plantarum</i>. This part of the project required us to assemble a Golden Gate compatible shuttle vector (that is replicable in both <i>E. coli</i> and <i>L. plantarum </i>) and transform <i>L. plantarum</i>.  Our experimental results are detailed below. </p>
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Revision as of 14:38, 12 October 2018


Results


There are millions of species of bacteria1, few of which are well characterized. However, these non-model organisms perform cellular processes and are native to harsh biomes, and this makes them of great interest to researchers2. Synthetic biologists take great interest in engineering such bacteria, in an attempt to harness their natural abilities3. Because finding the best way to genetically modify an organism can be a laborious and resource-demanding process, we have developed a kit of modular plasmids, designed to quickly test multiple broad host range origins of replication (ORIs). To do this, we used a cloning technique called Golden Gate Assembly to build plasmids. Golden Gate Assembly uses Type IIs restriction enzymes, which allows researchers to pick the overhang each cut produces. Therefore, many parts can be assembled together at once, in a specific order. The plasmids are minimal and standardized4. They include barcode sequences and reporter genes to indicate which plasmid is which. A mixture of all the plasmids is transformed into a single sample of host bacteria, and the colored reporters allow the researcher to quickly determine which ORIs allow the host to replicate the plasmid.