Difference between revisions of "Part:BBa K2315034"
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The HSL receiver LasR from <i>P.aeruginosa</i> activates expression of GFP protein in response to 3OC12HSL.  
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The AHL receiver LasR from <i>P.aeruginosa</i> activates expression of GFP protein in response to 3OC12-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:LasRpLasGFPPARTS.png|thumb|center|700px|<b>Fig. 1 Genetic Circuit Design</b>]]
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[[File:T--Shanghaitech--lasrfx.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 LasR will bind to the AHL molecule 3OC12HSL, dimerize and bind to the pLas regulatory sequence to activate GFP expression.
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When AHL is added with concentration higher than a critical value, the constitutively expressed LasR will bind to the AHL molecule 3OC12-HSL, dimerize and bind to the pLas regulatory sequence to activate GFP expression.
  
 
==<b>Fluorescent Response to cognate 3OC12-HSL</b>==
 
==<b>Fluorescent Response to cognate 3OC12-HSL</b>==
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To test this part, we used standard 3OC12HSL (HSL produced by LasI in P.aeruginosa) to determine the response curve.  
+
To test this part, we used standard 3OC12HSL (HSL produced by LasI in <i>P.aeruginosa</i>) to determine the response curve.  
  
 
[[File:T--Shanghaitech--LasRGFPLF.png|thumb|center|800px|<b>Fig. 1 LasR-pLas-GFP‘s response to cognate 3OC12HSL </b>]]
 
[[File:T--Shanghaitech--LasRGFPLF.png|thumb|center|800px|<b>Fig. 1 LasR-pLas-GFP‘s response to cognate 3OC12HSL </b>]]
  
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==Orthogonality test against non-cognate inducers==
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==<b>Orthogonality test against non-cognate inducers</b>==
 
We have characterized crosstalk response of LasR to several non-cognate AHLs:
 
We have characterized crosstalk response of LasR to several non-cognate AHLs:
 
[[File:T--Shanghaitech--LasRorthogonality.png|thumb|center|800px|<b>Fig. 2 Orthogonality test of LasR-pLas-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 ]]
 
[[File:T--Shanghaitech--LasRorthogonality.png|thumb|center|800px|<b>Fig. 2 Orthogonality test of LasR-pLas-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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Furthermore, we test it under fluorescence microscope. Fig. 3 shows four testing samples.
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[[File:T--Shanghaitech--2315034-3.png|thumb|center|700px|<b>Fig. 3 Orthogonality test under fluorescence microscope</b> <b> a) Interlab Study Test Device 1 <bbpart>BBa_J364000</bbpart> b) Las molecule 3OC12-HSL as inducer c) Tra molecule 3OC8-HSL as inducer d) Rpa molecule Coumaroyl-HSL as inducer</b>]]
  
 
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.
 
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.
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==<b>Usages in our Project</b>==
 
==<b>Usages in our Project</b>==
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AHL receiver from P.aeruginosa, actives expression of GFP protein.
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AHL receiver from <i>P.aeruginosa</i>, actives expression of GFP protein.
  
 
 

Latest revision as of 21:52, 1 November 2017


LasR-pLas-GFP HSL inducible fluorescent actuator

Group: Shanghaitech iGEM 2017


The AHL receiver LasR from P.aeruginosa activates expression of GFP protein in response to 3OC12-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 LasR will bind to the AHL molecule 3OC12-HSL, 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

Furthermore, we test it under fluorescence microscope. Fig. 3 shows four testing samples.

Fig. 3 Orthogonality test under fluorescence microscope a) Interlab Study Test Device 1 BBa_J364000 b) Las molecule 3OC12-HSL as inducer c) Tra molecule 3OC8-HSL as inducer d) Rpa molecule Coumaroyl-HSL as inducer

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.


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