Difference between revisions of "Part:BBa K3147004"

Revision as of 16:06, 14 October 2019

mRFP1 fused to a TEV cleavage site cleaved

I : parts BBa_K3147004 (mRFP1-TEVcs) function

The Montpellier 2019 team made this reporter gene construct in order to compare the basal fluorescence of mRFP1-TEV cutting site with the mRFP1-TEVcs-ssrA (BBa_ K3147003) construct. This construction produces a mRFP1 (BBA_ E1010) fused in C-ter to a TEV cutting site after cutting (ENLYFQ). This construction 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 mRFP1 with a "cleaved" TEV cutting site to a mRFP1 with a SSRA proteolysis tag separated with a TEV recognition site. We compared the basal fluorescence of the strain NEB10β of E. coli transformed with the mRFP1-TEVcs construction compared to E. coli NEB10β transformed with the mRFP1-TEVcs-SSRA construction. The construction was cloned by Gibson Assembly in a pBbE8K-RFP (https://www.addgene.org/35363/ ) backbone under the control of a BAD type promoter, in order to control its expression.

Figure 2: mRFP1-TEVcs-SRRA reporter gene in its pBbB8k-GFP backbone.

Fluorescence was quantified after arabinose induction at 1% by reading in a Plate Reader overnight. Here, we measurated the fluorescence of mRFP-TEVcs-SSRA and mRFP-TEVcs.

Figure 3 :Measurement of the fluorescence at 30°C and 37°C in RFU of bacteria expressing mRFP-TEVcs or mRFP-TEVcs-SSRA

Sequence and Features