Method

Promoter family

Fig. 1

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.2

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 designed reverse promoter, promoter isolation primer, that is to isolate randomized promoter by annealing downstream of it [Fig.4].

Fig.3

Fig.4

Assay

To measure transcription activities, we prepared two popular reporter genes and one antibiotics resistance gene, mRFP1, lacZ&alpha ;, and Kanamycin resistance gene.