Our modeling predictions were confirmed at the bench
Before beginning experiments, genome scale modeling and flux balance analysis were performed in order to maximize the success of our electron donor constructs in viable cells (GSM and FBA are further explained on our modeling page). By running these models, it was found that by increasing flux through the POR5 reaction pathway, more reduced flavodoxin/ferredoxin was produced, without seriously inhibiting cell growth. Also, the models showed that by knocking out a competing pathway, the PDH reaction pathway, it increases flux through the POR5 pathway, further increasing the amount of product made.
Using this information, our team researched genes necessary to create these reactions. The most vital gene for the POR5 reaction was pfo, which produces pyruvate flavodoxin/ferredoxin oxidoreductase that reduces the electron donors, giving us more usable product; this gene was joined with both the petF and fldA construct in the hopes of producing more electron donors in the cells. The most vital gene for the PDH reaction was ΔaceE; a strain of E. Coli with this gene knocked out was purchased and our electron donor constructs were inserted into this strain. In the end we had eight total constructs: fldA, fldA-pfo, petF, and petF-pfo both in DH10B (our wild type) and our knockout strain.
When these construct were tested with our biotin assay, our result supported the claims made by the models; not only did the fldA-pfo construct produce a higher amount of biotin (indirectly meaning more electron donors were produced) than just the fldA construct in DH10B, but also all 3 constructs tested produced more biotin in the knockout cells than what they produced in normal DH10B cells.
Overall, the use of modeling outside the wet lab allowed us to investigate a way to successfully improve the functions of our constructs inside viable cells.