Second, our introduction of genes PabA, PabB and PabC facilitates the production of enzymes that are responsible for the conversion of chorismate to pABA. Chorismate is transformed into para-aminobenzoic acid through chemical reactions involving the enzymes 4-amino-4-deoxychorismate (ADC) synthase and 4-amino-4-deoxychorismate (ADC) lyase, with the former encoded by PabA, and the latter encoded by PabB and PabC. Our approach was to introduce extra copies of genes PabA, PabB, and PabC into our cells. PabA and PabB together produce ADC synthase, which is responsible for the conversion of chorismate to 4-amino-4-deoxychorismate. Expression of PabC results in ADC lyase, the enzyme that would convert 4-amino-4deoxychorismate to pyruvate and our desired product, pABA.

Since pABA is an intermediate in the folic acid pathway, pABA will be consumed in the pathway’s downstream chemical reactions. Dihydropteroate (DHP) synthase catalyzes the reaction that converts pABA to 7,8-dihydropteroate, a precursor to 7,8-dihydrofolate [3]. By knocking out the gene that expresses dihydropteroate synthase, folate production is halted, thus preventing the conversion of pABA to subsequent folate precursors. To carry this out, we planned to use CRISPR-Cas9 for excision of the gene and to transform into E. coli. To verify that the cells were successfully transfected, cells were grown in medium with selection markers, such as chloramphenicol and folate. Afterwards, the growing cells would be replated on medium containing only the selection marker. Colonies that did not survive on the replated medium are successful, because they are folate-dependent since their folate pathway was disrupted by the DHP synthase knockout.

We also wanted to take into account previous work that has been done on pABA production. Previously, the ETH Zurich 2012 team [4] had produced a biobrick containing pabA and pabB from E. coli (BBa_K909014), but did not see large increase in the amount of pABA being produced. However, we also wanted a biobrick with pabC from E. coli in order to investigate whether or not the pabC enzyme was limiting the rate of pABA production. We also introduced used the DAHP synthase and Cas9 gene knockout to affect other parts of the Shikimate pathway besides just the steps involving chorismate to pABA.

To implement more copies of DAHP enzyme into E. coli, we used BBa_K1060000. When we performed CPCR, we had a product of band size ~1,000bp, as shown in lane 8 and by the blue arrow of Figure 1. According to the BioBrick page, the product should be 1,053 bp. This was promising; however when we sequenced it, the results did not overlap with the published sequence.