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.