Part:BBa_K2315034

LasR-pLas-GFP HSL inducible fluorescent actuator

LasR-pLas-GFP
Group: Shanghaitech iGEM 2017

The HSL receiver LasR 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

Fig. 1 Genetic Circuit Design

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

Fluorescent Response to cognate 3OC12-HSL

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

Fig. 1 LasR-pLas-GFP‘s response to cognate 3OC12HSL

Orthogonality test against non-cognate inducers

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

Fig. 2 Orthogonality test of LasR-pLas-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 LasR 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.

LasR-pLas-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
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 383
  Illegal AgeI site found at 580
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1700
  

AHL receiver from P.aeruginosa, actives expression of GFP protein.

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 NheI site found at 979
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 375
  Illegal AgeI site found at 572
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1740