Due to time constraint, the RT-PCR and GC-MS experiments on the different recombinant E. coli clones were performed only once, and will have to be repeated in future studies in order to verify the statistical significance of the data.

For overexpression studies of the Δ9-Δ12-Δ15 desaturase gene cluster in E. coli, we will repeat subcloning of the desaturase gene cluster into pSB1C3 to create a regulatable expression system for ALA production.

Attempts will be made to co-transform E. coli TOP10 cells with (1) the fadL-fadD expression cassette, (2) the ‘tesA expression cassette, and (3) the Δ9-Δ12-Δ15 desaturase gene cassette to construct a Fit Coli strain. Expression of the gene cassettes will be analysed by qRT-PCR, whilst fatty acid uptake and ALA production will be monitored by GC-MS analyses.

Because unsaturated fatty acids are expensive sources of carbon and energy from the perspective of a cell, and an ALA export protein may not be present in E. coli to facilitate ALA export for human uptake, we will consider a pH-induced autolysis system to facilitate extracellular ALA release. When bacteria reach a certain section of the gut of a particular pH, the cell will open up so the human gut may gain access to the useful ALA. Alternatively, a bile salt-inducible system to activate yet another engineered gene from E. coli, which encodes the MazF protein (a stable toxin) that initiates program death in the cells will also be tested.