Difference between revisions of "Part:BBa K3147004"

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<partinfo>BBa_K3147004 short</partinfo>
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<div align="center"><partinfo>BBa_K3147004 short</partinfo></div>
  
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===I : parts BBa_K3147004 (mRFP1-TEVcs) function===
 
  
  
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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.
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===I : parts BBa_K3147004 function===
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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 [[Part:BBa_ K3147000]]. This construction produces mRFP1 fused in C-ter to a sequence corresponding to a TEV cutting site after cleavage (ENLYFQ).
  
 
  <div align="center">[[File:design2K3147004.png|650px]]</div>
 
  <div align="center">[[File:design2K3147004.png|650px]]</div>
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===II. Proof of function===
 
===II. Proof of function===
  
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The experimental approach used to test the activity of these reporters was to compare the basal fluorescence rate of mRFP1 with a "cleaved" TEV cutting site to a mRFP1 with a SSRA proteolysis tag separated with a TEV recognition site.  We compared the basal fluorescence of the strain NEB10β of E. coli  transformed with the mRFP1-TEVcs construction compared to E. coli NEB10β transformed with the mRFP1-TEVcs-SSRA construction.  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.  
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This construction was cloned by Gibson Assembly in a pBbB8k (https://www.addgene.org/35363) backbone under the control of a pBAD promoter.
  
  
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[[File:PlasmideK3147004.png|400px]]
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<div align="center">[[File:PlasmideK3147004.png|400px]]</div>
  
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Figure 2: mRFP1-TEVcs-SRRA reporter gene in its pBbB8k-GFP backbone.
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<div align="center"><b>Figure 2</b>: mRFP1-TEVcs reporter gene in its pBbB8k-GFP backbone.</div>
  
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Fluorescence was quantified after arabinose induction at 1% by reading in a Plate Reader overnight. Here, we measurated the fluorescence of mRFP-TEVcs-SSRA and mRFP-TEVcs.
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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.
  
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[[File:resultK3147003.png|400px]]
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<div align="center">[[File:resultK3147003.png|400px]]</div>
  
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Figure 3 :Measurement of the fluorescence at 30°C and 37°C in RFU of bacteria expressing mRFP-TEVcs or mRFP-TEVcs-SSRA
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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-ssrA</div>
  
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==Reference==
  
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[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
  
 
<!-- Add more about the biology of this part here
 
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Latest revision as of 16:29, 20 October 2019


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 Part:BBa_ K3147000. This construction produces mRFP1 fused in C-ter to a sequence corresponding to a TEV cutting site after cleavage (ENLYFQ).

Design2K3147004.png
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.


PlasmideK3147004.png
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.

ResultK3147003.png
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]