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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HSL receiver from P.aeruginosa, actives 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 part is one of the interchangeable AHL receivers. Full collection could be viewed at:
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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>]]
  
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When proper AHL added with concentration higher than a critical value, The constitutively expressed LasR will bind the signaling AHL molecule, dimerize and bind to pLas regulatory sequence before GFP gene and actives its 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 3OC12HSL, 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 validate the part's design, we've used standard 3OC12HSL (HSL produced by LasI in P.aeruginosa) sample to determine the response curve of this part.
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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've characterized the crosstalk between LasR and 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