Method

Promoter family

As our first step for constructing original promoter family, we synthesized theoretically ideal consensus sequence to bind σ factor. This should ensure that promoter will form the most stable complex with σ factor. We synthesized such a consensus promoter showed in the figure above, originated from consensus sequence and lac operon promoter (pLac) (fig.1).

fig. 1　How to make consensus promoter. R means that you can choose anilline or glycine. D means that you can pick out from anillin or glycine, thymine.

We constructed consensus promoter by primer annealing (fig.2). For mutating hexamer at -35 region, a promoter randomize primer which has random hexamer (NNNNNN) at -35 region was used, but other sequence in the primer is same with consensus promoter (fig.3). We transformed the promoter-randomized constructs. We could logically find 4096 kinds of colonies on the plate. Then, we selected 10 promoters according to assay result. We designed promoter isolation primer, that is to isolate randomized promoter by annealing downstream of it (fig.4).

fig.2 Consensus promoter primer.

fig.3 Randomization at -35 region.

fig.4 Structure of promoter isolating from construct.

Assay

To measure transcription activities, we prepared two popular reporter genes and one antibiotics resistance gene, mRFP1, LacZα, and Kanamycin resistance gene.