Part:BBa_K3147001

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.

Figure 1 : Construct Design: sfGFP with cleaved TEV cutting site.

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.