Part:BBa_K2315014

RhlR-pRhl-GFP
Group: Shanghaitech iGEM 2017

The HSL receiver RhlR from P.aeruginosa activates expression of GFP protein in response to 3OC12HSL.

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

File:RhlRpRhlGFPPARTS.png

Fig. 1 Genetic Circuit Design

When AHL is added with concentration higher than a critical value, the constitutively expressed RhlR will bind to the AHL molecule 3OC12HSL, dimerize and bind to the pRhl regulatory sequence to activate GFP expression.

Fluorescent Response to cognate 3OC12-HSL

To test this part, we used standard 3OC12HSL (HSL produced by RhlI in P.aeruginosa) to determine the response curve.

File:T--Shanghaitech--RhlRGFPLF.png

Fig. 1 RhlR-pRhl-GFP‘s response to cognate 3OC12HSL

Orthogonality test against non-cognate inducers

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

File:T--Shanghaitech--RhlRorthogonality.png

Fig. 2 Orthogonality test of RhlR-pRhl-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 3OC12-HSL

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

Usages in our Project

We developed a measurement protocol using the fluorescent protein coupled AHL receiver germs to measure the actual AHLs concentration in high precision and sensitivity in compared with LC-MS.

pCon-RhlR-pRhl-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
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 301
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 776
  Illegal BsaI.rc site found at 1674