Part:BBa_K2315026

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

[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.

File:T--Shanghaitech--RpaRGFPLF.png

Fig. 1 RpaR-pRpa-GFP‘s response to cognate CoumaroylHSL

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NotI site found at 587
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1700
  Illegal SapI.rc site found at 500