Part:BBa_K3147004
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 691
- 1000COMPATIBLE WITH RFC[1000]
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