Difference between revisions of "Part:BBa K3147001"
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| − | The Montpellier 2019 team made this reporter gene construct in order to compare the basal fluorescence of sfGFP-TEV cutting site with the sfGFP-TEVcs-SRRA construct (BBa_ K3147000). This construction produces a sfGFP(bs)[1] (BBA_K1365020) fused in C-ter to a TEV cutting site after cutting (ENLYFQ). | + | The Montpellier 2019 team made this reporter gene construct in order to compare the basal fluorescence of sfGFP-TEV cutting site with the sfGFP-TEVcs-SRRA construct (BBa_ K3147000). This construction produces a sfGFP(bs)[1] (BBA_K1365020) fused in C-ter to a TEV cutting site after cutting (ENLYFQ). It can be used as a reporter gene. |
<div align="center">[[File:design2K31470001.png|650px]]</div> | <div align="center">[[File:design2K31470001.png|650px]]</div> | ||
Revision as of 15:13, 14 October 2019
I : parts BBa_K3147001 (Pc-sfGFP-TEVcs) function :
The Montpellier 2019 team made this reporter gene construct in order to compare the basal fluorescence of sfGFP-TEV cutting site with the sfGFP-TEVcs-SRRA construct (BBa_ K3147000). This construction produces a sfGFP(bs)[1] (BBA_K1365020) fused in C-ter to a TEV cutting site after cutting (ENLYFQ). It can be used as a reporter gene.
II. Proof of function
The experimental approach used to test the activity of these reporters was to compare the basal fluorescence rate of the sfGFP with a TEV cutting site "cleaved" . That's why we made a construction: sfGFP-TEVcs similar by removing the proteolysis tag, and simulating a cut by the TEV. We compared the basal fluorescence of strain E. coli NEB10β transformed with the construction type sfGFP-TEVcs compared to E. coli NEB10β transformed with the construction sfGFP-TEVcs-SSRA. Fluorescence was quantified by reading with a Plate Reader overnight. We also expressed this part in the pBbE8K-RFP backbone (https://www.addgene.org/35327/) under a pBAD promoter. The cloning was made by Gibson Assembly.
Figure 2: sfGFP-TEVcs reporter gene in its backbone pBbE8K-RFP.
Figure 3: Measurement of the fluorescence at 30°C and 37°C in RFU of bacteria expressing sfGFP-TEVcs or sfGFP-TEVcs-SSRA
We compared the basal fluorescence of the E. coli strain NEB10β transformed with the sfGFP-TEVcs construction and the E. coli NEB10β transformed with the sfGFP-TEVcs-SSRA construction. Fluorescence was quantified after induction with arabinose concentrated at 1% by reading with Plates Reader all night. Here, we measured the fluorescence of the sfGFP-TEVcs-SSRA at 30 and 37°C and sfGFP-TEVcs.