Difference between revisions of "Part:BBa K2315014"

Revision as of 21:32, 1 November 2017

RhlR-pRhl-GFP
Group: Shanghaitech iGEM 2017

The HSL receiver RhlR 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 C4-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 RhlR will bind to the AHL molecule C4-HSL, dimerize and bind to the pRhl regulatory sequence to activate GFP expression.

Fluorescent Response to cognate C4--HSL

To test this part, we used standard C4-HSL (HSL produced by RhlI in P.aeruginosa) to determine the response curve.

File:T--Shanghaitech--RhlRGFPLF.png

Fig. 1 RhlR-pRhl-GFP‘s response to cognate C4-HSL

Orthogonality test against non-cognate inducers

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

File:T--Shanghaitech--RhlRorthogonality.png

Fig. 2 Orthogonality test of RhlR-pRhl-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 C4--HSL

It has shown that RhlR 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.

pCon-RhlR-pRhl-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 301
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 776
Illegal BsaI.rc site found at 1674

@@ Line 8: / Line 8: @@
 ==<b>Usage and Biology</b>==
-[[File:RhlRpRhlGFPPARTS.png|thumb|center|700px|<b>Fig. 1 Genetic Circuit Design</b>]]
+[[File:T--Shanghaitech--rhlfx.jpg|thumb|center|700px|<b>Fig. 1 Genetic Circuit Design</b>]]
 When AHL is added with concentration higher than a critical value, the constitutively expressed RhlR will bind to the AHL molecule C4-HSL, dimerize and bind to the pRhl regulatory sequence to activate GFP expression.