Difference between revisions of "Part:BBa K3147000"

(→‎I : parts BBa_K3147000 (Pc-sfGFP-TEVcs-SSRA) function)
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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 gene. In the presence of TEV the ssrA is separated and allows the sfGFP to glow without being degraded.  
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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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<div align="center"><b>Figure 1 </b>: Construct Design: sfGFP fused to an ssrA proteolysis tag with a TEV cutting site between them. </div>
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<div align="center"><b>Figure 1 </b>: Construct Design: sfGFP fused to an ssrA proteolysis tag with a TEV cutting site in between. </div>
  
 
===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 the sfGFP with a TEV recognition site "cleaved" to this construction. To do so, we made a control construction: sfGFP-TEVcs(BBa_K3147001) similar to this one by removing the proteolysis tag and simulating a cut by the TEV.
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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.
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We expressed this part in a plasmid with an arabinose promoter : pBbE8K-RFP backbone (https://www.addgene.org/35327/). We cloned it by Gibson Assembly.
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<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 quantified  after induction with arabinose concentrated at 1% with the plate reader all night.
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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.
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Here are the fluorescence of the sfGFP-TEVcs-ssrA and the  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.
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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.  
  
 
<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]