Difference between revisions of "Part:BBa K2315006"

Revision as of 20:11, 1 November 2017

RpaR-pRpa-GFP
Group: Shanghaitech iGEM 2017

The HSL receiver RpaR from Rhodopseudomonas palustris (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

File:RpaRpRpaGFPPARTS.png

Fig. 1 Genetic Circuit Design

When AHL is added with 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 no obvious crosstalk with other HSLs.

RpaR-RHOPA-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
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 493
Illegal XhoI site found at 128
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 136
Illegal NgoMIV site found at 200
Illegal NgoMIV site found at 485
Illegal NgoMIV site found at 573
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1704

Revision as of 14:05, 1 November 2017 (view source) Luofang (Talk \| contribs) ← Older edit		Revision as of 20:11, 1 November 2017 (view source) Luofang (Talk \| contribs) Newer edit →
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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 ]]		[[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 ]]

−	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~~.	+	It has shown that RpaR is sensitive to it's cognate HSL and has no obvious crosstalk with other HSLs.