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New Idea - A PROTOCOL FOR ENGINEERING PROMOTERS TO FIT THE DESIGN SPECIFICATIONS Motive Constructing a functional genetic circuit requires assembling genetic devices and getting them to work together. The main challenge in genetic circuit design lies in selecting well-matched genetic components that when coupled, reliably produce the desired behavior. Although the parameter values are calculated by model equations, it is hard to select the biological part that reliably implements a desired cellular function with quantitative values. In general, to control gene expression is achieved by placing the gene of interest under the control of an inducible promoter. Strong and inducible promoters are widely used in recombinant protein over-expression. However, many current application in synthetic biology, particular those to metabolic optimization and control analysis, require the capacity to precisely tune the levels of gene expression. To overcome this problem, the promoters which can control the expression of downstream genes are necessary. Our team designs a simple and rapid protocol to generate a promoter library. This promoter library with different transcriptional strength can be built to tune the specific parameter values that model equations indicated. A strategy is applied in our protocol. Degenerated primers designed for PCR are used to generate mutations in promoter regions. The promoter activity can be assayed using a reporter protein (GFP). Because the reporter protein activity has positive correlation to promoter activity, we can choose the suitable transcriptional strength of the promoter for our design scheme.

Principle The original observation that the regions flanking the ?35 (TTGACA) and ?10 (TATAAT) bacterial promoter consensus sequences affect promoter strength is the blueprint for the tuning of promoter activity. If random mutations are created at the ?35 and ?10 sequences, then such promoters will represent many variations in promoter strength (Fig. 1). <a href="http://2009.igem.org/Image:Newprot1.png" ><img src="http://2009.igem.org/wiki/images/0/06/Newprot1.png" border="0"></a> Fig. 1. Degenerated primers designed for PCR are used to generate mutations in promoter regions. The library of promoters is created where the -35 or -10 sequences are amplified by degenerated primers with some random mutations (red star), then such a library can represent many variations in promoter strength.