Difference between revisions of "Part:BBa K3147004"

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  <div align="center"><b>Figure 3</b> :Measurement of the fluorescence at 30°C and 37°C of bacteria expressing mRFP-TEVcs or mRFP-TEVcs-SSRAin RFU</div>
 
  <div align="center"><b>Figure 3</b> :Measurement of the fluorescence at 30°C and 37°C of bacteria expressing mRFP-TEVcs or mRFP-TEVcs-SSRAin RFU</div>
  
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==Reference==
  
 
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Revision as of 16:11, 14 October 2019


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 compare the basal fluorescence of mRFP1-TEV cutting site with the mRFP1-TEVcs-ssrA (BBa_ K3147003) construct. This construction produces a mRFP1 (BBA_ E1010) fused in C-ter to a TEV cutting site after cutting (ENLYFQ). This construction can be used as a reporter gene.

Design2K3147004.png
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.


PlasmideK3147004.png
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.

ResultK3147003.png
Figure 3 :Measurement of the fluorescence at 30°C and 37°C of bacteria expressing mRFP-TEVcs or mRFP-TEVcs-SSRAin RFU

Reference

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]