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] [2] [3] (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 this construction to the fluorescence of the sfGFP-TEVcs-ssrA (BBa_K3147000). That's why we made two constructions similar by removing the proteolysis tag from one and simulating a cut by the TEV.

We 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.

We compared the basal fluorescence of strain E. coli NEB10β transformed with the construction type sfGFP-TEVcs to an E. coli NEB10β transformed with the construction sfGFP-TEVcs-ssrA. Fluorescence was quantified by measuring after induction with arabinose concentrated at 1% it with the plate reader overnight. Here are the fluorescences obtained from the sfGFP-TEVcs-SSRA and sfGFP-TEVcs at 30 and 37°C.

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

Reference

[1] McGinness, Baker, Sauer. 2006. Mol. Cell. 22:701.

[2] Overkamp, W. et al. (2013) Benchmarking various green fluorescent protein variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for live cell imaging. Appl. About. Microbiol. 79: 6481-6490

[3] Sarah Guiziou et al. 2016. “A part toolbox to tune genetic expression in Bacillus subtilis” Nucleic Acids Research, 2016, Vol. 44, No. 15 7495–7508.