Davidson Missouri W/PLac

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Background

pLac in a natural environment transcribes three adjacent genes which add up to approximately 5000nt. pLac is stimulated by a metabolite of lactose: allolactose (or an analog of allolactose: IPTG). IPTG induces transcription by binding to LacI. LacI binds to the one of three lac operators (o1) which is located near the start of transcription and overlaps into RNAP's interaction site at the -10 pribnow box. Several other factors of control of the lac operon have been identified such as the system's reliance on the cAMP-CAP complex, and repression by DNA looping between operator sequences.
Lacoperon.png

CAP and the UV5 mutation

Catabolite activator protein (CAP) binds to the lac operon 60 bases upstream of the start of transcription. Together with cAMP, the CAP-cAMP complex is though to recruit RNAP to its -35 and -10 consensus sequences. The UV5 double mutation at the -10 consensus sequence makes the operon independent of the CAP binding region. While pLac derived biobrick promoters have side stepped the issue of RNAP recruiting by inducing with IPTG, we incorporated the UV5 mutation into our version of part BBa_R0011. We synethsized the hybrid promoter by annealing oligonucleotides using Davidson's software().