Future Work

Testing 4-Enzyme Design

Now that we have successfully inserted the POAP pathway into the minimal cell, we need to test it! We'll be using GC-MS screens to detect and quantify levels of pathway intermediates. Mutagenesis will be used to ensure proper functionality of each enzyme in the pathway.

Measure Carbon Fixation

To determine the success of the POAP cycle in Syn3.0b, we will quantify how much carbon the POAP pathway sequesters in vivo. In vitro, at 50C under anaerobic conditions, the POAP pathway fixes 8.0 nmol CO2 min–1 mg–1 CO2-fixing enzymes [1]. This rate is expected to be slightly lower in vivo, but still more efficient than the Calvin cycle.

Developing a Minimal Media

SP4 media, the current media we grow the cells in, is very expensive, costing about $1000 per liter [2]. This is not sustainable for large-scale growth and production of these cells. To tackle this problem, we plan to develop a minimal media that will allow large-scale growth at a cheaper rate.

Addressing the Oxalate Byproduct

One byproduct of the POAP cycle is the molecule Oxalate. Unfortunately, oxalate is not very useful to the cell and may even be toxic to the cells meaning this byproduct needs to be addressed to ensure healthy growth of cells, especially for large-scale growth. Potential ideas include incorporating a pathway into the cell that uses oxalate or precipitating the oxalate out as calcium oxalate.

Sources

[1] https://pubs.acs.org/doi/abs/10.1021/acscatal.1c04151

[2] https://hardydiagnostics.com/u86

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