Difference between revisions of "Part:BBa K3147003"

(→‎I : parts BBa_K3147003 (mRFP1-TEVcs-SSRA) function)
(→‎II. Proof of function)
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===II. Proof of function===
 
===II. Proof of function===
  
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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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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 pBAD promoter.
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  <div align="center">[[File:PlasmideK3147003.png|400px]]</div>
 
  <div align="center">[[File:PlasmideK3147003.png|400px]]</div>
  
 
  <div align="center"><b>Figure 2</b>: mRFP1-TEVcs-ssrA reporter gene in its pBbB8k-GFP backbone.</div>
 
  <div align="center"><b>Figure 2</b>: mRFP1-TEVcs-ssrA reporter gene in its pBbB8k-GFP backbone.</div>
  
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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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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 overnight induction with 1% arabinose on a plate reader.  
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Below are the fluorescence measurements of the mRFP1-TEVcs-ssrA and of the mRFP1-TEVcss at 30 and 37°C.  
  
  

Revision as of 09:45, 18 October 2019


mRFP1 fused to a TEV-cleavable ssrA tag

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

This construction produces an mRFP1 (BBA_E1010)[1] 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 is a sister construction to our sfGFP TEV reporter (Ba_K3147000). This construction was used to test the specificity of KARMA using a VHH targeting GFP. In the presence of TEV the ssrA is cleaved and mRFP1 is not degraded anymore.


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 pBAD promoter.

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 overnight induction with 1% arabinose on a plate reader.

Below are the fluorescence measurements of the mRFP1-TEVcs-ssrA and of the mRFP1-TEVcss at 30 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 in 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

[2] Sunohara, T., Abo, T., Inada, T., & Aiba, H. (2002). The C-terminal amino acid sequence of nascent peptide is a major determinant of SsrA tagging at all three stop codons. RNA (New York, N.Y.), 8(11), 1416–1427. doi:10.1017/s1355838202020198


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]