Part:BBa_K3147004

mRFP1 fused to a TEV cleavage site cleaved

I : parts BBa_K3147004 function

The Montpellier 2019 team made this reporter gene construct in order to obtain a positive control for TEV mediated proteolysis of the ssrA tag fused the mRFP1 reporter (BBa_ K3147000). This construction produces mRFP1 fused in C-ter to a sequence corresponding to a TEV cutting site after cleavage (ENLYFQ).

Figure 1 : Construct Design: mRFP1 with TEV cutting site cleaved.

II. Proof of function

This construction was cloned by Gibson Assembly in a pBbB8k (https://www.addgene.org/35363) backbone under the control of a pBAD promoter.

Figure 2: mRFP1-TEVcs reporter gene in its pBbB8k-GFP backbone.

We compared the basal fluorescence of the strain NEB10β of E. coli transformed with the mRFP1-TEVcs construction to an E. coli NEB10β strain transformed with the mRFP1-TEVcs-ssrA construction. Fluorescence was quantified after arabinose induction at 1% with a plate reader overnight. Here are the fluorescence of mRFP-TEVcs-ssrA and mRFP-TEVcs at 30°C and 37°C.

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

Reference

[1] Levraud, Jean-Pierre et al. 2007. « Identification of the Zebrafish IFN Receptor: Implications for the Origin of the Vertebrate IFN System ». The Journal of Immunology 178(7): 4385‑94. doi:10.4049/jimmunol.178.7.4385

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 691
  
• 1000
  COMPATIBLE WITH RFC[1000]