Team:UCSF/Project/Conjugation/Promoter

After our initial promoter assays our goal was to create engineered versions of our promoters responsive to high and low levels of inducer, and here we’ve chosen pLAC promoter as proof of concept.

The lactose inducible promoter pLAC, first discovered in Escherichia coli and serving as a core component of lac operon responsible for lactose metabolism, is one of the best-studied and engineered prokaryotic transcriptional regulatory system. The switch like behavior of this promoter in response to lactose level is achieved through interaction of a regulatory protein LacI and small fragments of DNA sequence, named lac operators, within the promoter region.

The LacI protein monomers self-associate into an unusual tetramer that appears roughly as a V-shaped dimer of dimers (See Figure 1A for detailed information), and could be further divided into four discrete functional units: a N-terminal headpiece with a helix-turn-helix motif capable of binding to the DNA, a hinge region connecting headpiece with core, and the protein core with a N-terminal lactose binding domain and a C-terminal helix responsible for dimerization. Each dimeric repressor is capable of binding to a 21-base-pair duplex deoxyoligonucleotides, the lac operator site. (Wilson et al., 2007; Lewis, 2011) Meanwhile, the lac operator sequence is a pseudo-symmetric DNA sequence that was first identified to be a 27-base-pair section (lacO1) (Gilbert et al., 1973) and further narrowed down to around 17 base pairs for minimal specific binding requirement (Bahl et al., 1977) (Figure 1B).