Difference between revisions of "Part:BBa K3147003"

(→‎I : parts BBa_K3147003 (mRFP1-TEVcs-SSRA) function :)
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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  TEV cutting site "cleaved". For this purpose we made a control construction: mRFP1-TEVcs (BBa_K3147004) similar to it by removing the proteolysis tag, and simulating a cut by the TEV. We compared the basal fluorescence of strain E. coli NEB10β transformed with the mRFP1-TEVcs construction compared to E. coli NEB10β transformed with the mRFP1-TEVcs-SSRA construction. Fluorescence was quantified after induction with arabinose concentrated at 1% by reading with a Plate Reader overnight. The construction was cloned by Gibson Assembly in a pBbB8k-GFP (https://www.addgene.org/35363/ ) backbone under the control of a BAD type promoter, in order to control its expression.
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The experimental approach to test the activity of these reporters was to compare the basal fluorescence rate of mRFP1 with a  TEV cutting site "cleaved" to this construction. For this purpose we made a control construction: mRFP1-TEVcs (BBa_K3147004) similar to this one by removing the proteolysis tag and simulating a cut by the TEV. The construction was cloned by Gibson Assembly in a pBbB8k-GFP (https://www.addgene.org/35363/ ) backbone under the control of a BAD type promoter, in order to control its expression.
  
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[[File:PlasmideK3147003.png|400px]]
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<div align="center">[[File:PlasmideK3147003.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-ssrA reporter gene in its pBbB8k-GFP backbone.</div>
  
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[[File:resultK3147003.png|500px]]
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We compared the basal fluorescence of strain E. coli NEB10β transformed with the mRFP1-TEVcs construction to an E. coli NEB10β strain transformed with the mRFP1-TEVcs-ssrA construction. Fluorescence was quantified after induction with arabinose concentrated at 1% with the plate reader overnight. Here are the fluorescence of mRFP-TEVcs-ssrA and mRFP-TEVcs at 30°C and 37°C. We can see that the ssrA tag is reducing the fluorescence from the mRFP-TEVcs.
  
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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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Here, we measurated the fluorescence of mRFP-TEVcs-SSRA and mRFP-TEVcs. We can see that the SSRA tag is reducing the fluorescence from the mRFP-TEVcs.
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<div align="center">[[File:resultK3147003.png|500px]]</div>
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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 in RFU</div>
  
 
==Reference==
 
==Reference==

Revision as of 15:59, 14 October 2019


mRFP1 fused to a TEV-cleavable ssrA tag

I : parts BBa_K3147003 (mRFP1-TEVcs-SSRA) function :

The Montpellier 2019 team made a reporter gene construction in order to carry out their proof of concept. This construction produces an mRFP1 (BBA_E1010) fused in C-ter with a fast degradation tag called ssrA [2] (BBA_M0050). The TEV cutting site (BBa_J18918) was added between the mRFP1 and the ssrA tag. This construction can be used as a reporter gene. In the presence of TEV the ssrA is separated and allows the mRFP to glow without being degraded.

DesignK3147003.png
Figure 1: Construct Design: mRFP1 fused to an SSRA proteolysis tag with a TEV cutting site between the two.

II. Proof of function

The experimental approach to test the activity of these reporters was to compare the basal fluorescence rate of mRFP1 with a TEV cutting site "cleaved" to this construction. For this purpose we made a control construction: mRFP1-TEVcs (BBa_K3147004) similar to this one by removing the proteolysis tag and simulating a cut by the TEV. The construction was cloned by Gibson Assembly in a pBbB8k-GFP (https://www.addgene.org/35363/ ) backbone under the control of a BAD type promoter, in order to control its expression.

PlasmideK3147003.png
Figure 2: mRFP1-TEVcs-ssrA reporter gene in its pBbB8k-GFP backbone.

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


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

Reference

add ref


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]