Difference between revisions of "Part:BBa K3147001"

(→‎I : parts BBa_K3147001 (Pc-sfGFP-TEVcs) function :)
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===I : parts BBa_K3147001 (Pc-sfGFP-TEVcs) function :===
 
  
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__NOTOC__
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<div align="center"><partinfo>BBa_K3147001 short</partinfo></div>
  
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The Montpellier 2019 team made this reporter gene construct in order to compare the basal fluorescence of sfGFP-TEV cutting site with the sfGFP-TEVcs-SRRA construct (BBa_ K3147000). This construction produces a sfGFP(bs)[1] (BBA_K1365020) fused in C-ter to a TEV cutting site after cutting (ENLYFQ).  
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===I : parts BBa_K3147001 function===
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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).
  
 
  <div align="center">[[File:design2K31470001.png|650px]]</div>
 
  <div align="center">[[File:design2K31470001.png|650px]]</div>
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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 the sfGFP with a TEV cutting site  "cleaved" . That's why we made a  construction: sfGFP-TEVcs similar by removing the proteolysis tag, and simulating a cut by the TEV.  We compared the basal fluorescence of strain E. coli NEB10β transformed with the construction type sfGFP-TEVcs compared to E. coli NEB10β transformed with the construction sfGFP-TEVcs-SSRA. Fluorescence was quantified by reading with a Plate Reader overnight. We also 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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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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[[File:PlasmideK3147001.png|400px]]
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<div align="center">[[File:PlasmideK3147001.png|400px]]</div>
  
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Figure 2: sfGFP-TEVcs reporter gene in its backbone pBbE8K-RFP.
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<div align="center"><b>Figure 2</b>: sfGFP-TEVcs reporter gene in its backbone pBbE8K-RFP.</div>
  
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[[File:resultK3147001.png|500px]]
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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 (BBa_K3147000). Fluorescence was measured on a plate reader after overnight induction with 1% arabinose.
  
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Figure 3: Measurement of the fluorescence at 30°C and 37°C in RFU of bacteria expressing sfGFP-TEVcs or sfGFP-TEVcs-SSRA
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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. 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.
  
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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% by reading with Plates Reader all night.
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  <div align="center">[[File:resultK3147001.png|500px]]</div>
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Here, we measured the fluorescence of the sfGFP-TEVcs-SSRA at 30 and 37°C and sfGFP-TEVcs.
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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==
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[1] McGinness, Baker, Sauer. 2006. Mol. Cell. 22:701.
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[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
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[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.
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3147001 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K3147001 parameters</partinfo>
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<!-- -->

Latest revision as of 06:51, 20 October 2019


sfGFP fused to a TEV cleavage site cleaved


I : parts BBa_K3147001 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

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]