Team:Baltimore BioCrew/Results

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Results

• Summary of Results






• Future Work

Summary of Results

B. Theta Genes

Our goal was to help repopulate the gut microbiome after antibiotics. During our research, we discovered Mucous Associated Functional Factors (MAFF) proteins, proteins synthesized by B. theta, that support the symbiosis of many bacterial species within the gut and maintain the health of the epithelial cell lining. As B. theta is anaerobic, difficult to grow in a lab, and is extremely sensitive (thus often wiped out of the gut after antibiotics), we decided that a MAFF supplement would be the best solution to the problem. Thus, we aimed to synthesize the MAFF proteins in E. coli by creating two plasmids (K3301012 and K3301013) each capable of expressing one of two MAFF proteins. The two genes for the proteins are BT2268 and BT2269, which we used to develop the basic parts BBa_K3301001 and BBa_K3301002.

During our work, we found it difficult to synthesize the plasmid containing BT2268. Due to its length, we ordered the gene in two parts that would be later linked by a BamHI restriction site. However, the star activity and lack of inactivation temperature for this enzyme made the gene impossible to ligate reliably into one piece, and thus all transformation efforts failed. As a result, we focused our energy on BT2269.

Here are our results: After digestion, ligation, and transformation, positive BT2269 colonies grew. After extracting and digesting plasmids from the colonies with EcoRI and PstI, a gel was run. On it, we saw a band of about 2kbp, the size we expected, thus indicating that a successful uptake of the BT2269 gene and mechanisms to produce it into Ecoli.

Plasmids were digested with EcoRI and PstI and run on a gel. A band of about 2kbp was displayed, signaling a correct transformation.

Killswitch

After disappointing results while testing the original killswitch, we focused our attention to the redesigned version. After some trouble with synthesis due to the complexity of the construct, we eventually got our DNA shipped after we changed out the terminator for something less complex. A positive gel electropheresis told us that our DNA was as long as it should be. After colonies were grown successfully, we proceeded to run a new experiment.

This data clearly follows a visible trend. There is an immediate jump in the OD of each sample, save one, as the xylose concentration goes above 0%, as now the bacteria is expressing the antitoxin. After that initial jump, the reading start to even out, with some OD readings growing. This tells us that the initial jump is around the xylose concentration needed to maintain a colony.

Future Work

We aim to continue to expand upon this project by
-Purifying the MAFF protein from the positive colonies
-Ensuring the reproducibility of our data
-Finding a new method of ligating BTT268 so we can synthesize it