Difference between revisions of "Part:BBa K3147001"

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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 this construction to the fluorescence of the sfGFP-TEVcs-ssrA (BBa_K3147000). That's why we made two constructions similar by removing the proteolysis tag from one and simulating a cut by the TEV.
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We expressed this part in the pBbE8K backbone (https://www.addgene.org/35327) under a pBAD promoter. The cloning was made by Gibson Assembly.  
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We expressed this part in the pBbE8K-RFP backbone (https://www.addgene.org/35327) under a pBAD promoter. The cloning was made by Gibson Assembly.  
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  <div align="center">[[File:PlasmideK3147001.png|400px]]</div>
 
  <div align="center">[[File:PlasmideK3147001.png|400px]]</div>
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  <div align="center"><b>Figure 2</b>: sfGFP-TEVcs reporter gene in its backbone pBbE8K-RFP.</div>
 
  <div align="center"><b>Figure 2</b>: sfGFP-TEVcs reporter gene in its backbone pBbE8K-RFP.</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 E. coli strain NEB10β transformed with the sfGFP-TEVcs construction and the E. coli NEB10β transformed with the sfGFP-TEVcs-ssrA construction (BBa_K3147000). 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. Our part simulating cleavage effectively has a strong fluorescence.  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. Our part simulating cleavage effectively has a strong fluorescence.  We can see that the ssrA system is more efficient at 37C as described in part M0050 characterization.  

Revision as of 09:34, 18 October 2019

I : parts BBa_K3147001 (Pc-sfGFP-TEVcs) function

The Montpellier 2019 team made this reporter gene construct in order to obtain a positive control for TEV mediated proteolysis of the ssrA tag fused the sfGFP reporter (BBa_ K3147000). This construction produce sfGFP(bs)[1] [2] [3] (BBA_K1365020) fused in C-ter to a sequence corresponding to a TEV cutting site after cleavage (ENLYFQ).

Design2K31470001.png
Figure 1 : Construct Design: sfGFP with cleaved TEV cutting site.

II. Proof of function

We expressed this part in the pBbE8K backbone (https://www.addgene.org/35327) under a pBAD promoter. The cloning was made by Gibson Assembly.

PlasmideK3147001.png
Figure 2: sfGFP-TEVcs reporter gene in its backbone pBbE8K-RFP.

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 (BBa_K3147000). 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. Our part simulating cleavage effectively has a strong fluorescence. We can see that the ssrA system is more efficient at 37C as described in part M0050 characterization.

ResultK3147001.png
Figure 3: Measurement of the fluorescence at 30°C and 37°C of bacteria expressing sfGFP-TEVcs or sfGFP-TEVcs-SSRA in GFP/OD

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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 947
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]