Difference between revisions of "Part:BBa K2315026"

Revision as of 21:37, 1 November 2017

RpaR-pRpa-GFP
Group: Shanghaitech iGEM 2017

The HSL receiver RpaR from Sphingobium sp. EP60837 (strain ATCC BAA-98 / CGA009) activates expression of GFP protein in response to Coumaroyl-HSL.

This Receiver can be easily replaced by other AHL receivers in our collection. A full collection could be found in: http://2017.igem.org/Team:Shanghaitech/Library

Usage and Biology

thumb|center|700px|Fig. 1 Genetic Circuit Design

When AHL is added with a concentration higher than a critical value, the constitutively expressed RpaR will bind to the AHL molecule Coumaroyl-HSL, dimerize and bind to the pRpa regulatory sequence to activate GFP expression.

Fluorescent Response to cognate Coumaroyl-HSL

To test this part, we used standard Coumaroyl-HSL (HSL produced by RpaI in P.aeruginosa) to determine the response curve.

Orthogonality test against non-cognate inducers

We have characterized crosstalk response of RpaR to several non-cognate AHLs:

Fig. 2 Orthogonality test of RpaR-pRpa-GFP (i):Fluorescent response to cognate and non-cognate AHLs (ii)Dose-Response curves for cognate and non-cognate AHLs (iii-vi)Fluorescent response to non-cognate AHLs in compared with Coumaroyl-HSL

It has shown that RpaR is sensitive to it's cognate HSL and has obvious crosstalk with 3OC8-HSL in Tra System in relatively high concentration.

pCon-RpaR-Sphingobium sp. EP60837-pRpa-GFP

pCon-RpaR-Sphingobium sp. EP60837-pRpa-GFP

Sequence and Features