Difference between revisions of "Part:BBa K2315011"
(→Orthogonality test against non-cognate inducers) |
|||
| (3 intermediate revisions by the same user not shown) | |||
| Line 2: | Line 2: | ||
Group: <b>Shanghaitech iGEM 2017</b> | Group: <b>Shanghaitech iGEM 2017</b> | ||
| − | The HSL receiver LasR from | + | 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: | + | 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 |
==<b>Usage and Biology</b>== | ==<b>Usage and Biology</b>== | ||
| Line 16: | Line 16: | ||
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. | ||
| − | + | [[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== | ||
We have characterized crosstalk response of LasR to several non-cognate AHLs: | We have characterized crosstalk response of LasR to several non-cognate AHLs: | ||
| − | [[File:T--Shanghaitech-- | + | [[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 ]] |
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. | 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. | ||
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
Contents
Usage and Biology
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.
Orthogonality test against non-cognate inducers
We have characterized crosstalk response of LasR to several non-cognate AHLs:
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 383
Illegal AgeI site found at 580 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1700