GC-MS Data

Simplified diagram of GC-MS setup. [1]

Goal

Confirm the functionality of the POAP pathway via gas chromatography-mass spectrometry (GC-MS) analysis.

Approach

To better troubleshoot any issues, we divided the POAP cycle into two halves such that they produce and consume pyruvate and acetate in a complimentary fashion. Thus, we can compare results to simple and clear predictions of how the cycle should work.

Two-enzyme plasmid design. [2]

Predicted Results

This table organizes all of our predicted results for the minimal cell grown at 37ºC and 43.7ºC, with and without halves of the POAP cycle. Due to time and scheduling constraints, we could not test for the full POAP cycle transformed into the minimal cell under either temperature condition (see *). Below is the legend for abbreviations and symbols.

WT: wild-type minimal cell without any genetic modification and grows at 37ºC

TALE: wild-type minimal cell that is thermally adapted to grow at 43.7ºC

A1: minimal cell with the ACS-PFOR enzymes (one half of the POAP cycle)

B1: minimal cell with the PYC-OAH enzymes (one half of the POAP cycle)

- : decrease + : increase ++: significant increase ~ : about the same

Metabolite Testing

We tested for acetate and pyruvate because they are intermediates of the POAP cycle, and they fall along the cycle halves we created for cycle functionality analysis. We also tested for lactic acid because pyruvate is quickly converted into lactic acid under anaerobic conditions. Lastly, we tested for oxaloacetate (the final product of the POAP cycle) because it was expected to be produced in the PYC-OAH half of the cycle. However, it was not detected in any sample we prepared, so no data can be presented for it.

Acetate Quantification

The acetate concentration (µM) was normalized to the cell count since an acceptable standard curve was created. Acetate was only tested for in the supernatant. We did not expect it to be used elsewhere and assumed it would diffuse out of the cell.

Pyruvate Detection

The pyruvate concentration was normalized to the ion count (m/z = 174) per cell (a standard curve was not prepared). Pyruvate was expected to remain in the cell pellet (P), but it was also found in the supernatant (S).

Lactic Acid Detection

The lactic acid concentration was normalized to the ion count (m/z = 147) per cell (a standard curve was not prepared). Lactic was expected to remain in the cell pellet (P), but it was also found in the supernatant (S).

Conclusions & Future Work

Some of the actual results do not match our predicted results, which indicates a need for further troubleshooting and testing. Potential reasons for these differences include errors in sample preparation, the rest of the enzymes besides PFOR not having optimal activity at 43.7ºC, and decreased expression of some of the enzymes.

Future work consists of a variety of tasks. One task involves optimizing the activity for all enzymes in the cycle, which includes determining the optimal gene order for the enzymes on the plasmid (genes that appear later in the plasmid tend to have reduced expression) and figuring out one cell growth temperature that works for all four enzymes. Another task is conducting GC-MS analysis on all sample types previously done plus the four-enzyme construct transformed into the WT and TALE minimal cell. During this run, biological and technical replicates are recommended to produce statistically significant results. Additionally, robust standard curves for all metabolites being tested should be prepared to properly quantify the concentration of each metabolite per cell.

MACS Logo. [3]

Sources

[1] https://measurlabs.com/methods/gas-chromatography-mass-spectrometry-gc-ms/

[2] Created by the 2023-2024 MACS Senior Design Team.

[3] Created by the 2023-2024 MACS Senior Design Team.

Table was produced in Google Sheets.

Metabolite graphs were produced in Microsoft Excel.

Made By: Jamie Gatus

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