Difference between revisions of "Part:BBa K3147000"

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__NOTOC__
 
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<partinfo>BBa_K3147000 short</partinfo>
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<div align="center"><partinfo>BBa_K3147000 short</partinfo></div>
  
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===I : parts BBa_K3147000 (Pc-sfGFP-TEVcs-SSRA) function===
 
  
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The Montpellier 2019 team submitted a reporter gene construction in order to carry out their proof of concept. This construction produces an sfGFP(bs) [1] [2] [3] (BBA_K1365020) merged into C-ter with a fast degradation tag called ssrA [4] [5]  (BBA_M0050). The TEV cutting site (BBa_J18918) has been added between the sfGFP and the ssrA tag. This construction can be used as a reporter for proteolysis activity by TEV. In the presence of TEV the ssrA is cleaved and sfGFP is not degraded anymore.  
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===I : parts BBa_K3147000 function===
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The Montpellier 2019 team submitted a reporter gene construction in order to carry out their proof of concept. This construction produces an sfGFP(bs) [1] [2] [3] [[Part:BBa_K1365020]] fused in C-terminal with a fast degradation tag called ssrA [4] [5]  [[Part:BBa_M0050]]. The TEV cutting site [[Part:BBa_J18918]] has been added between the sfGFP and the ssrA tag. This construction can be used as a reporter for proteolysis activity by TEV. In the presence of TEV the ssrA is cleaved and sfGFP is not degraded anymore.  
  
 
<div align="center">[[File:Pc-sfGFP-TEVcs-SSRA2.png|650px]]</div>
 
<div align="center">[[File:Pc-sfGFP-TEVcs-SSRA2.png|650px]]</div>
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===II. Proof of function===
 
===II. Proof of function===
  
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As a positive control, we built a construct simulating a ssrA cleavage by the TEV, by expressing sfGFP with a C-terminal sequence corresponding to the cleaved TEV recognition (BBa_K3147001 ). We expressed this part in a plasmid under the control of the arabinose promoter: pBbE8K-RFP backbone (https://www.addgene.org/35327/). We cloned it by Gibson Assembly.
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As a positive control, we built a construct simulating a ssrA cleavage by the TEV, by expressing sfGFP with a C-terminal sequence corresponding to the cleaved TEV recognition [[Part:BBa_K3147001]]. We expressed this part in a plasmid under the control of the arabinose promoter: pBbE8K-RFP backbone (https://www.addgene.org/35327/). We cloned it by Gibson Assembly.
  
 
<div align="center">[[File:pbad-pc-sfGFP-TEVcs-SRRA.png|350px]]
 
<div align="center">[[File:pbad-pc-sfGFP-TEVcs-SRRA.png|350px]]
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<div align="center"><b>Figure 2</b>: sfGFP-TEVcs-ssrA reporter gene in its pBbE8K-RFP backbone.</div>
 
<div align="center"><b>Figure 2</b>: sfGFP-TEVcs-ssrA reporter gene in its pBbE8K-RFP backbone.</div>
  
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We compared the basal fluorescence of the E. coli strain NEB10β transformed with the sfGFP-TEVcs construction and the E. coli NEB10β transformed with the sfGFP-TEVcs-ssrA construction. Fluorescence was measured on a plate reader after overnight induction with 1% arabinose.
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We compared the basal fluorescence of the <i>E. coli</i> strain NEB10β transformed with the sfGFP-TEVcs construction and the <i>E. coli</i> NEB10β transformed with the sfGFP-TEVcs-ssrA construction. Fluorescence was measured on a plate reader after overnight induction with 1% arabinose.
  
 
Below are the fluorescence measurements of the sfGFP-TEVcs-ssrA and of the sfGFP-TEVcs at 30 and 37°C. We can see that the ssrA tag is causing a lot of degradation of the protein. We can see that the ssrA system is more efficient at 37C as described in part M0050 characterization.  
 
Below are the fluorescence measurements of the sfGFP-TEVcs-ssrA and of the sfGFP-TEVcs at 30 and 37°C. We can see that the ssrA tag is causing a lot of degradation of the protein. We can see that the ssrA system is more efficient at 37C as described in part M0050 characterization.  
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<div align="center">[[File:résultat K314700.png|700px]]</div>
 
<div align="center">[[File:résultat K314700.png|700px]]</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 sfGFP-TEVcs or sfGFP-TEVcs-ssrA in RFU</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 sfGFP-TEVcs or sfGFP-TEVcs-ssrA</div>
  
 
== Reference: ==
 
== Reference: ==

Latest revision as of 16:21, 20 October 2019


sfGFP fuse to TEV-cleavable ssrA tag


I : parts BBa_K3147000 function

The Montpellier 2019 team submitted a reporter gene construction in order to carry out their proof of concept. This construction produces an sfGFP(bs) [1] [2] [3] Part:BBa_K1365020 fused in C-terminal with a fast degradation tag called ssrA [4] [5] Part:BBa_M0050. The TEV cutting site Part:BBa_J18918 has been added between the sfGFP and the ssrA tag. This construction can be used as a reporter for proteolysis activity by TEV. In the presence of TEV the ssrA is cleaved and sfGFP is not degraded anymore.

Pc-sfGFP-TEVcs-SSRA2.png
Figure 1 : Construct Design: sfGFP fused to an ssrA proteolysis tag with a TEV cutting site in between.

II. Proof of function

As a positive control, we built a construct simulating a ssrA cleavage by the TEV, by expressing sfGFP with a C-terminal sequence corresponding to the cleaved TEV recognition Part:BBa_K3147001. We expressed this part in a plasmid under the control of the arabinose promoter: pBbE8K-RFP backbone (https://www.addgene.org/35327/). We cloned it by Gibson Assembly.

Pbad-pc-sfGFP-TEVcs-SRRA.png
Figure 2: sfGFP-TEVcs-ssrA reporter gene in its pBbE8K-RFP backbone.

We compared the basal fluorescence of the E. coli strain NEB10β transformed with the sfGFP-TEVcs construction and the E. coli NEB10β transformed with the sfGFP-TEVcs-ssrA construction. Fluorescence was measured on a plate reader after overnight induction with 1% arabinose.

Below are the fluorescence measurements of the sfGFP-TEVcs-ssrA and of the sfGFP-TEVcs at 30 and 37°C. We can see that the ssrA tag is causing a lot of degradation of the protein. We can see that the ssrA system is more efficient at 37C as described in part M0050 characterization.

Résultat K314700.png
Figure 3:Measurement of the fluorescence at 30°C and 37°C of bacteria expressing sfGFP-TEVcs or sfGFP-TEVcs-ssrA

Reference:

[1] McGinness, Baker, Sauer. 2006. Mol. Cell. 22:701.

[2] Overkamp, W. et al. (2013) Benchmarking various green fluorescent protein variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for live cell imaging. Appl. About. Microbiol. 79: 6481-6490

[3] Sarah Guiziou et al. 2016. “A part toolbox to tune genetic expression in Bacillus subtilis” Nucleic Acids Research, 2016, Vol. 44, No. 15 7495–7508.

[4] Fernandez-Rodriguez, Jesus, et Christopher A. Voigt. 2016. « Post-TranslationalControl of Genetic Circuits Using Potyvirus Proteases ». Nucleic Acids Research 44(13): 6493‑6502.

[5] 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
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 41
    Illegal NheI site found at 53
    Illegal NheI site found at 76
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 82
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]