Difference between revisions of "Part:BBa K3147003"

Revision as of 15:53, 14 October 2019

mRFP1 fused to a TEV-cleavable ssrA tag

I : parts BBa_K3147003 (mRFP1-TEVcs-SSRA) function :

The Montpellier 2019 team made a reporter gene construction in order to carry out their proof of concept. This construction produces an mRFP1 (BBA_E1010) fused in C-ter with a fast degradation tag called ssrA [2] (BBA_M0050). The TEV cutting site (BBa_J18918) was added between the mRFP1 and the ssrA tag. This construction can be used as a reporter gene. In the presence of TEV the ssrA is separated and allows the mRFP to glow without being degraded.

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 TEV cutting site "cleaved". For this purpose we made a control construction: mRFP1-TEVcs (BBa_K3147004) similar to it 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 mRFP1-TEVcs construction compared to E. coli NEB10β transformed with the mRFP1-TEVcs-SSRA construction. Fluorescence was quantified after induction with arabinose concentrated at 1% by reading with a Plate Reader overnight. The construction was cloned by Gibson Assembly in a pBbB8k-GFP (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.

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

Here, we measurated the fluorescence of mRFP-TEVcs-SSRA and mRFP-TEVcs. We can see that the SSRA tag is reducing the fluorescence from the mRFP-TEVcs.

Reference

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Sequence and Features