Reporter

Part:BBa_K3147001

Designed by: Thomas Bessede   Group: iGEM19_Montpellier   (2019-10-12)
Revision as of 09:21, 18 October 2019 by Jbonnet (Talk | contribs) (→‎II. Proof of function)

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.

Design2K31470001.png
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.

PlasmideK3147001.png
Figure 2: sfGFP-TEVcs reporter gene in its backbone pBbE8K-RFP.

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 measured on a plate reader after overnight induction with 1% arabinose.

Below are the fluorescence measurements of the sfGFP-TEVcs-ssrA and of the sfGFP-TEVcs at 30 and 37°C. We can see that the ssrA tag is causing a lot of degradation of the protein. Our part simulating cleavage effectively has a strong fluorescence. We can see that the ssrA system is more efficient at 37C as described in part M0050 characterization.

ResultK3147001.png
Figure 3: Measurement of the fluorescence at 30°C and 37°C of bacteria expressing sfGFP-TEVcs or sfGFP-TEVcs-SSRA in GFP/OD

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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 947
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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