Difference between revisions of "Part:BBa K3147003"
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===I : parts BBa_K3147003 (mRFP1-TEVcs-SSRA) function===
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===I : parts BBa_K3147003 function===
  
 
This construction produces an mRFP1 (BBA_E1010)[1] fused in C-ter with a ssrA fast degradation tag  [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.  
 
This construction produces an mRFP1 (BBA_E1010)[1] fused in C-ter with a ssrA fast degradation tag  [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.  
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  <div align="center">[[File:designK3147003.png|650px]]</div>
 
  <div align="center">[[File:designK3147003.png|650px]]</div>
  
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  <div align="center"><b>Figure 1</b>: Construct Design: mRFP1 fused to an SSRA proteolysis tag with a TEV cutting site between the two.</div>
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  <div align="center"><b>Figure 1</b>: Construct Design: mRFP1 fused to an ssrA proteolysis tag with a TEV cutting site between the two.</div>
  
 
===II. Proof of function===
 
===II. Proof of function===

Revision as of 14:40, 20 October 2019


mRFP1 fused to a TEV-cleavable ssrA tag


I : parts BBa_K3147003 function

This construction produces an mRFP1 (BBA_E1010)[1] fused in C-ter with a ssrA fast degradation tag [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

This 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
Stats30CK3147003.png
Figure 4: Statistical analysis of the effect of ssrA tag on the mRFP-TEVcs at 30°C
Stats37CK3147003.png
Figure 5: Statistical analysis of the effect of ssrA tag on the mRFP-TEVcs at 37°C

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]