Part:BBa_K3147001

I : parts BBa_K3147001 (Pc-sfGFP-TEVcs) function :

The Montpellier 2019 team made this reporter gene construct in order to be able to compare the basal fluorescence 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).

Figure 1: Construct Design: sfGFP with cleaved TEV cutting site. The objective of the construction was to be able to compare the basal fluorescence of the cleaved sfGFP-TEV cutting site, in comparison with the reporter gene sfGFP-TEVcs-SSRA (BBa_ K3147000).

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.