To observe a decrease in TMA concentration, it is necessary to have an initial time point, or reference point. At zero hours, there should be almost all TMA and no TMAO. With four hours as our endpoint, the two-hour interval serves as a midway point. With these three data points, we should observe a decrease in TMA as time increases and an increase in TMAO.

For all samples, 100 µL of 1 g/L of TMA was mixed with 400 µL of the probiotic solution suspended in LB broth. This established a baseline of known TMA concentration per sample for every single sample. The remaining 400 µL was of the probiotic solution.

The controls of any experiment are vital to understanding what factors are unaccounted for and possible bias an experiment. The following eight controls are what we prepared to ensure that our experiment will behave as expected.

For Dr. Wood's experimentation and for our own HPLC experimentation, we adopted similar controls.

Experimental Controls

• Concentrated TMA at 6.65 M
• Dilute TMA of at ~1 g/L
• LB Broth and TMA (Non dilute), with 25 mL LB to 5 mL TMA
• LB Broth and TMA (Dilute), with 25 mL LB to 5 mL TMA
• LB Broth and Probiotic 200 2 mL solution
• LB Broth and Probiotic 201 2 mL solution
• LB Broth and Probiotic 202 2 mL solution
• LB Broth

These controls should demonstrate no change in TMA, no reported TMA concentration, or ensure there is no contamination of our samples and that the detection found on HPLC or MS-MS is actually TMA.

The TMA Oxidation Assay Developed with Dr. Arthur "Carl" Whittington was designed with a well-plate, ensuring efficient workflow in the lab and minimizing waste. This approach allows us to run many trials of our experiment concurrently. Due to the changing procedures to troubleshoot TMA-FMOC detection as well as quantification, this protocol is suspect to change. The final protocol to demonstrate the efficacy of our protocols will be released during the Wiki Thaw when we overcome difficulties in the laboratory.

For Dr. Wood's team, the team utilized the constant ratio of 100 µL of 1 g/L of TMA to 400 µL of the probiotic solution. Every single sample for all the enzymes was ran at 37 degrees Celsius and was incubated at zero hours, two hours, and four hours. To understand how TMA-oxidation is affected by oxygen levels, we tested these samples at ambient conditions with a normal oxygen levels, or roughly 21%. We then performed these tests in incubators with 5% CO2, providing a lower concentration of oxygen. This was important to understand because the small intestine has lowered oxygen levels and we were concerned with how this would affect our enzymes' performance. Every single sample was made in duplicate to ensure that any results we get are not occurring by chance.

Twenty-eight samples of all enzymes at both ambient and low oxygen conditions utilized a 0.22-micron nylon filtration for removing cells. This is because the cells interfere with absorbance values in HPLC, making it difficult to interpret TMA concentration levels. Just in case the nylon fitration removed TMA or caused other unforseen error, nine samples of the E. coli with roseovarius species 217 enzyme at both ambient and lowered oxygen conditions were centrifuged. Centrifugation allowed us to aliquot only the supernatant and put this in a 1.5 mL tube for analysis, leaving the cells behind. What is most important about this system of removing the cells is that it mitigates the error involved with analysis. As our E. coli respires, it will continuously produce the enzyme of interest. If we send our samples off for analysis with cells, the cells will likely continue to respire and therefore change the actual levels of TMA and TMAO, no longer yielding data for the time points of zero hours, two hours, and four hours. By removing these cells, we prevented this error from affecting our results.

The above tube rack contains the final samples sent off to Aurora, Colorado for testing: 45 samples with eight controls and 37 tests of each enzyme over a span of zero hours, two hours, and four hours. We are currently waiting for results to come in.

The following PDF explains all of our protocols used for transformation, LB broth preparation, and all fundamental work not particular to the project.