Team:PKU Beijing/Parts Characterization/BBa K228009

BBa K228009 - AraC protein(reversed sequence) and Pbad promoter

Designed by Rengcheng Gao Group: iGEM09_PKU_Beijing (2009-09-18)
Input: Arabinose molecules
Output: GFP fluorescence

Description

We have constructed the inducement system composed of L-Arabinose inducible Pbad promoter and constitutively expressed AraC regulatory protein. When arabinose is absent, the AraC protein binds to and represses Pbad very efficiently. Thus, the transcription of any downstream coding gene is restricted. On the other hand, the import of arabinose could change the conformation of AraC protein, successfully prevent it repressing Pbad promoter and activate the transcription of downstream coding gene, such as GFP.

This part is constructed for two purposes. First, we coupled two parts, an AraC protein pand a Pbad promoter, in order to characterize the latter one (Part ) and obtain more information about this promoter. We place a GFP coding gene(Part ) downstream of Pbad promoter to construct a report system. If arabinose is added to the culture, the expression of GFP will be triggered and green fluorescence can be tested. We have tested the function of this promoter according to concentration gradient and time scale, as is shown below. Secondly, we use this part as a sensor in our AND gate system, which works very efficiently.

The direction of AraC coding sequence is opposite compared to the Part . We have PCRed with primers which include standard enzyme-cutting sites. The aim of this reversion is to avoid unexpected expression leaking of downstream sequences.

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