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 be able to compare the basal fluorescence of mRFP1-TEV cutting site with the mRFP1-TEVcs-SRRA (BBa_ K3147003) construct. This construction produces a mRFP1 (BBA_ E1010) fused in C-ter to a TEV cutting site after cutting (ENLYFQ).  
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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).
  
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[[File:design2K3147004.png|650px]]
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<div align="center">[[File:design2K3147004.png|650px]]</div>
  
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Figure 1 : Construct Design:  mRFP1 with  TEV cutting site cleaved. The objective of the construction was to be able to compare the basal fluorescence of mRFP1 with the TEV cutting site cutted off, in comparison with the reporter gene mRFP1-TEVcs-SSRA  (BBa_ K3147003).
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<div align="center"><b>Figure 1</b> : Construct Design:  mRFP1 with  TEV cutting site cleaved. </div>
  
 
===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
 
<!-- Add more about the biology of this part here

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]