Part:BBa_K3147004

mRFP1 fused to a TEV cleavage site cleaved

I : parts BBa_K3147004 (mRFP1-TEVcs) 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

The experimental approach used to test the activity of these reporters was to compare the basal fluorescence rate of this construction to a mRFP1 fused to a ssrA proteolysis tag separated with a TEV recognition site. The construction was cloned by Gibson Assembly in a pBbE8K-RFP (https://www.addgene.org/35363/ ) backbone under the control of a BAD type promoter in order to control its expression.

Figure 2: mRFP1-TEVcs-SRRA 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-SSRAin RFU

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]