Difference between revisions of "Part:BBa K2315026"

 
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<b>RpaR-pRpa-GFP</b><br>
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Group: <b>Shanghaitech iGEM 2017</b>
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The HSL receiver RpaR from Sphingobium sp. EP60837 (strain ATCC BAA-98 / CGA009) activates expression of GFP protein in response to Coumaroyl-HSL.
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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>Usage and Biology</b>==
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[[File:T--Shanghaitech--rparfx.jpg|thumb|center|700px|<b>Fig. 1 Genetic Circuit Design</b>]]
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When AHL is added with a concentration higher than a critical value, the constitutively expressed RpaR will bind to the AHL molecule Coumaroyl-HSL, dimerize and bind to the pRpa regulatory sequence to activate GFP expression.
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==<b>Fluorescent Response to cognate Coumaroyl-HSL</b>==
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To test this part, we used standard Coumaroyl-HSL (HSL produced by RpaI in P.aeruginosa) to determine the response curve.
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[[File:T--Shanghaitech--RpaRGFPLF.png|thumb|center|800px|<b>Fig. 1 RpaR-pRpa-GFP‘s response to cognate CoumaroylHSL </b>]]
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==Orthogonality test against non-cognate inducers==
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We have characterized crosstalk response of RpaR to several non-cognate AHLs:
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[[File:T--Shanghaitech--RpaRorthogonality.png|thumb|center|800px|<b>Fig. 2 Orthogonality test of RpaR-pRpa-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 Coumaroyl-HSL ]]
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It has shown that RpaR 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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Latest revision as of 21:37, 1 November 2017

RpaR-pRpa-GFP
Group: Shanghaitech iGEM 2017

The HSL receiver RpaR from Sphingobium sp. EP60837 (strain ATCC BAA-98 / CGA009) activates expression of GFP protein in response to Coumaroyl-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 a concentration higher than a critical value, the constitutively expressed RpaR will bind to the AHL molecule Coumaroyl-HSL, dimerize and bind to the pRpa regulatory sequence to activate GFP expression.

Fluorescent Response to cognate Coumaroyl-HSL

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

File:T--Shanghaitech--RpaRGFPLF.png
Fig. 1 RpaR-pRpa-GFP‘s response to cognate CoumaroylHSL

Orthogonality test against non-cognate inducers

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

Fig. 2 Orthogonality test of RpaR-pRpa-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 Coumaroyl-HSL

It has shown that RpaR is sensitive to it's cognate HSL and has obvious crosstalk with 3OC8-HSL in Tra System in relatively high concentration.


pCon-RpaR-Sphingobium sp. EP60837-pRpa-GFP

pCon-RpaR-Sphingobium sp. EP60837-pRpa-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
    Illegal NotI site found at 587
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1700
    Illegal SapI.rc site found at 500