Breakdown of Trimethylamine via Trimethylamine Dehydrogenase to Minimize Heart Disease Caused by Red Meat Consumption

Phase 1: Arabinose induction

Figure 1. pBAD-D4 in DH5a E.coli, the negative control, on Endo agar plates at different concentrations of TMA

Conclusion:
The first result (on the left) shows very dark bands in the 0.001-0.1% range. This blot was detected using anti-HA with a purpose to detect the expression of TMADH. With this in mind, it is clear that the TMADH samples are highly expressed whenever 0.001-0.1% arabinose is added for induction. There is some indication of degradation in this sample as there are multiple bands. The largest band is approximately the correct predicted size of our protein, so we can still draw the conclusion that our protein was induced properly.
The second result (on the right) shows dark and clear bands within the 0.01-0.1% arabinose range. This blot was detected using anti-FLAG antibodies with a purpose to detect the expression of FDH instead. Clearly, the protein is being optimally expressed when 0.01-0.1% arabinose is added for induction.
Based on the results of this experiment, the concentration of arabinose that will be used to induce both pBAD-TMADH and pBAD-FDH expressing bacteria will be 0.05%.

Phase 2: TMADH Plate Assay

Figure 2. pBAD-D4 in DH5a E.coli, the negative control, on Endo agar plates at different concentrations of TMA

Figure 3. TMADH in DH5a E.coli, test group, on Endo agar plates at different concentrations of TMA

Conclusion:
These results are promising because before this point the bacteria have either not been able to grow on our plate conditions (and thus we adjusted the conditions between each trial) or there is no color change on the plate. However, in this trial, there is a significant color change- a noteworthy sample is the 0.01% TMA pBAD - TMADH plate which (if flipped to the other side) has bacteria with a metallic green sheen - a sign of high secretion of aldehydes. However, this is still not a perfect result as some of the BAD samples also show darker areas around the colonies although they shouldn’t as the DH5a cells shouldn’t be able to produce aldehydes due to the mutation on their LacC operon. Further, since instead of plating the same concentration of cells (we plated the same volume) the survival rate cannot be used as a result either. Since the negative control samples also exhibited significant color change we cannot make a definite conclusion based on this evidence.

Explanations and future studies:
All outliers or unpredicted results of this experiment can most likely be attributed to the many uncontrolled variables in the test environment. When the plates were incubated they had to be placed in a bag in order to contain the smell of TMA (smells like rotten fish) which introduce unpredicted variability in the humidity and temperature between each plate. Additionally, the reactant in endo agar tends to be relatively harsh and contributes to slower proliferation of bacteria, negatively impacting the accuracy of the results.
In future studies, cleaner assay methods could be used to test the expression of TMADH in bacteria that allow for more control over the test environment. One method that was suggested to us was mass spectrometry. As highlighted in the chemical breakdown of TMA by TMADH, a dimer known as dimethylamine (DMA) is a by product of the breakdown of TMA. Mass spectrometry detects the concentration of chemicals by ionizing compounds and measuring the emissions of the excited electrons. Because of the chemical structure of DMA, it would be feasible to measure its concentration using mass spectrometry. Therefore, in future studies it may be helpful to measure the formation of DMA in order to quantify the expression and effectiveness of our protein.

Phase 2: FDH Plate Assay

Figure 4. This is the result of the cell culture of bacteria +/- FDH on LB agar plates on a serial dilution of formaldehyde.

Figure 5. This is a graph comparing the growth rate of bacteria +/- FDH expression

Equation 1. This was the equation that we used to calculate survival/growth rate

Conclusion:
As illustrated in the diagram above, the survival rate of bacteria expressing FDH is significantly higher than the negative control bacteria without FDH. This shows that our FDH construct is successfully transformed into bacteria and thus can function properly in our test conditions and in our assay conditions.

Phase 3

Conclusion:
The grey box highlights one example of the region that was highly mutated. In this specific case, there is a high concentration of cytosine to thymine mutations. In order to achieve this result, we initially determined the correct PCR conditions for TMADH to perform a successful PCR. We also had to change the mutator to attach to the DNA polymerase from manganese ion to dPTP to increase the concentration of mutations that we had. Comparing -dPTP (without mutations) sequence to the +dPTP (with mutagenic analog) sequence, we can see a clear difference in base pair frequency.

Future studies:
In future studies, an appropriate assay would be used to screen the different mutations for the optimal one and then the process would be repeated over several generations to achieve a better enzyme. However, since the assay that we utilized in this project was not efficient enough, we were unable to begin the first round of screening.

Characterization

We characterized the Cornell 2017 part RBS-glutathione independent formaldehyde dehydrogenase, part number BBa_K2296013. Cornell’s original use of the part was to test the performance of the enzyme after putting the CDS of formaldehyde dehydrogenase (FDH) under an RBS. In our project, we used the same enzyme as the team, however, we also added other aspects of our part (such as the FLAG tag, STREP-tag, etc) in order to be optimized for the experiments that we performed. We characterized this part from the Cornell team in our project using a formaldehyde-LB culture assay.
In order to enrich the existing data on FDH we measured the survival rate of DH5a E. coli transformed with a plasmid secreting FDH. In our experiments, we wanted to prove that expression of FDH would increase the survival rate on LB agar plates with formaldehyde, thus, proving that the enzyme is being secreted effectively. Other results involving FDH, besides results involving the vector such as arabinose induction, are all part of characterization of this enzyme but are not detailed in this section. For more details refer to sections involving pBAD-FDH.
Our results showed that the survival rate of bacteria expression this glutathione independent FDH is higher than the negative control bacteria in nutrient environments that also contain formaldehyde: