Difference between revisions of "Part:BBa K2315014"
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Group: <b>Shanghaitech iGEM 2017</b>
 
Group: <b>Shanghaitech iGEM 2017</b>
  
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The HSL receiver RhlR from P.aeruginosa activates expression of GFP protein in response to 3OC12HSL.  
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The HSL receiver RhlR from Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) activates expression of GFP protein in response to C4-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
 
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
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==<b>Usage and Biology</b>==
 
==<b>Usage and Biology</b>==
  
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[[File:RhlRpRhlGFPPARTS.png|thumb|center|700px|<b>Fig. 1 Genetic Circuit Design</b>]]
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[[File:T--Shanghaitech--rhlfx.jpg|thumb|center|700px|<b>Fig. 1 Genetic Circuit Design</b>]]
  
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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.
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When AHL is added with concentration higher than a critical value, the constitutively expressed RhlR will bind to the AHL molecule C4-HSL, dimerize and bind to the pRhl regulatory sequence to activate GFP expression.
  
  
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==<b>Fluorescent Response to cognate 3OC12-HSL</b>==
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==<b>Fluorescent Response to cognate C4--HSL</b>==
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To test this part, we used standard 3OC12HSL (HSL produced by RhlI in P.aeruginosa) to determine the response curve.  
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To test this part, we used standard C4-HSL (HSL produced by RhlI in P.aeruginosa) to determine the response curve.  
  
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[[File:T--Shanghaitech--RhlRGFPLF.png|thumb|center|800px|<b>Fig. 1 RhlR-pRhl-GFP‘s response to cognate 3OC12HSL </b>]]
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[[File:T--Shanghaitech--rhlrflfx.jpg|thumb|center|800px|<b>Fig. 1 RhlR-pRhl-GFP‘s response to cognate C4-HSL </b>]]
  
 
==Orthogonality test against non-cognate inducers==
 
==Orthogonality test against non-cognate inducers==
 
We have characterized crosstalk response of RhlR to several non-cognate AHLs:
 
We have characterized crosstalk response of RhlR to several non-cognate AHLs:
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[[File:T--Shanghaitech--RhlRorthogonality.png|thumb|center|800px|<b>Fig. 2 Orthogonality test of RhlR-pRhl-GFP </b> (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 ]]
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[[File:T--Shanghaitech--RhlRorthogonality.png|thumb|center|800px|<b>Fig. 2 Orthogonality test of RhlR-pRhl-GFP </b> (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 C4--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.
 
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.

Latest revision as of 21:53, 1 November 2017

RhlR-pRhl-GFP
Group: Shanghaitech iGEM 2017

The HSL receiver RhlR from Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) activates expression of GFP protein in response to C4-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 concentration higher than a critical value, the constitutively expressed RhlR will bind to the AHL molecule C4-HSL, dimerize and bind to the pRhl regulatory sequence to activate GFP expression.


Fluorescent Response to cognate C4--HSL

To test this part, we used standard C4-HSL (HSL produced by RhlI in P.aeruginosa) to determine the response curve.

Fig. 1 RhlR-pRhl-GFP‘s response to cognate C4-HSL

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 C4--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