Difference between revisions of "Part:BBa K3147001"

(→‎I : parts BBa_K3147001 (Pc-sfGFP-TEVcs) function :)
(→‎II. Proof of function)
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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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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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[[File:PlasmideK3147001.png|400px]]
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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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Figure 2: sfGFP-TEVcs reporter gene in its backbone pBbE8K-RFP.
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<div align="center">[[File:PlasmideK3147001.png|400px]]</div>
  
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[[File:resultK3147001.png|500px]]
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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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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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We compared the basal fluorescence of strain E. coli NEB10β transformed with the construction type sfGFP-TEVcs to an E. coli NEB10β transformed with the construction sfGFP-TEVcs-ssrA. Fluorescence was quantified by measuring after induction with arabinose concentrated at 1% it with the plate reader overnight.
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Here are the fluorescences obtained from the sfGFP-TEVcs-SSRA and sfGFP-TEVcs at 30 and 37°C.
  
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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 in RFU </div>
  
 
==Reference==
 
==Reference==

Revision as of 15:38, 14 October 2019

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

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). It can be used as a reporter gene.

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

II. Proof of function

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.

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.

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

We compared the basal fluorescence of strain E. coli NEB10β transformed with the construction type sfGFP-TEVcs to an E. coli NEB10β transformed with the construction sfGFP-TEVcs-ssrA. Fluorescence was quantified by measuring after induction with arabinose concentrated at 1% it with the plate reader overnight. Here are the fluorescences obtained from the sfGFP-TEVcs-SSRA and sfGFP-TEVcs at 30 and 37°C.

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

Reference