Difference between revisions of "Part:BBa K2315011"

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=== LasR-GFP ===
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<b>LasR-pLas-GFP</b><br>
 
Group: <b>Shanghaitech iGEM 2017</b>
 
Group: <b>Shanghaitech iGEM 2017</b>
  
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The HSL receiver LasR from P.aeruginosa activates expression of GFP protein in response to 3OC12HSL.  
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The HSL receiver LasR 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 3OC12HSL.  
  
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This Receiver can be easily replaced by other AHL receivers in our collection. A full collection could be found in: (link tobe added!!!!!!!!)
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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>Introduction</b>==
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==<b>Usage and Biology</b>==
  
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(Figure1-mechanism to be added)
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[[File:LasRpLasGFPPARTS.png|thumb|center|700px|<b>Fig. 1 Genetic Circuit Design</b>]]
  
 
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.
 
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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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 P.aeruginosa) to determine the response curve.  
  
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(Figure2&3-response to 3OC12HSL to be added)
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[[File:T--Shanghaitech--LasRGFPLF.png|thumb|center|800px|<b>Fig. 1 LasR-pLas-GFP‘s response to cognate 3OC12HSL </b>]]
  
 
==Orthogonality test against non-cognate inducers==
 
==Orthogonality test against non-cognate inducers==
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We have characterized crosstalk response of LasR to several non-cognate AHLs
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We have characterized crosstalk response of LasR to several non-cognate AHLs:
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(Figure4-7-response to non-cognate inducers to be added)
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[[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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==Usage: Measuring unknown AHL production==
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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>==
 
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.
 
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.
  
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<partinfo>BBa_K2315011 short</partinfo>
 
<partinfo>BBa_K2315011 short</partinfo>
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
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===Usage and Biology===
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Latest revision as of 13:54, 1 November 2017

LasR-pLas-GFP
Group: Shanghaitech iGEM 2017

The HSL receiver LasR 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 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