Difference between revisions of "Part:BBa K3147004"

Revision as of 16:43, 13 October 2019

mRFP1 fused to a TEV cleavage site cleaved

I : parts BBa_K3147004 (mRFP1-TEVcs) function :

The Montpellier 2019 team made this reporter gene construct in order to be able to compare the basal fluorescence of mRFP1-TEV cutting site with the mRFP1-TEVcs-SRRA (BBa_ K3147003) construct. This construction produces a mRFP1 (BBA_ E1010) fused in C-ter to a TEV cutting site after cutting (ENLYFQ).

Figure 1 : Construct Design: mRFP1 with TEV cutting site cleaved. The objective of the construction was to be able to compare the basal fluorescence of mRFP1 with the TEV cutting site cutted off, in comparison with the reporter gene mRFP1-TEVcs-SSRA (BBa_ K3147003).

II. Proof of function

The experimental approach used to test the activity of these reporters was to compare the basal fluorescence rate of mRFP1 with a "cleaved" TEV cutting site to a mRFP1 with a SSRA proteolysis tag separated with a TEV recognition site. We compared the basal fluorescence of the strain NEB10β of E. coli transformed with the mRFP1-TEVcs construction compared to E. coli NEB10β transformed with the mRFP1-TEVcs-SSRA construction. The construction was cloned by Gibson Assembly in a pBbE8K-RFP (https://www.addgene.org/35363/ ) backbone under the control of a BAD type promoter, in order to control its expression.

Figure 2: mRFP1-TEVcs-SRRA reporter gene in its pBbB8k-GFP backbone.

Fluorescence was quantified after arabinose induction at 1% by reading in a Plate Reader overnight. Here, we measurated the fluorescence of mRFP-TEVcs-SSRA and mRFP-TEVcs.

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

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 691
1000
COMPATIBLE WITH RFC[1000]

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 __NOTOC__
 <partinfo>BBa_K3147004 short</partinfo>
+===I : parts BBa_K3147004 (mRFP1-TEVcs) function :===
 The Montpellier 2019 team made this reporter gene construct in order to be able to compare the basal fluorescence of mRFP1-TEV cutting site with the mRFP1-TEVcs-SRRA (BBa_ K3147003) construct. This construction produces a mRFP1 (BBA_ E1010) fused in C-ter to a TEV cutting site after cutting (ENLYFQ).
-add image
+[[File:designK3147004.png|650px]]
 Figure 1 : Construct Design:  mRFP1 with  TEV cutting site cleaved. The objective of the construction was to be able to compare the basal fluorescence of mRFP1 with the TEV cutting site cutted off, in comparison with the reporter gene mRFP1-TEVcs-SSRA  (BBa_ K3147003).
+===II. Proof of function===
+The experimental approach used to test the activity of these reporters was to compare the basal fluorescence rate of mRFP1 with a "cleaved" TEV cutting site to a mRFP1 with a SSRA proteolysis tag separated with a TEV recognition site.  We compared the basal fluorescence of the strain NEB10β of E. coli  transformed with the mRFP1-TEVcs construction compared to E. coli NEB10β transformed with the mRFP1-TEVcs-SSRA construction.  The construction was cloned by Gibson Assembly in a pBbE8K-RFP (https://www.addgene.org/35363/ ) backbone under the control of a BAD type promoter, in order to control its expression.
+[[File:PlasmideK3147004.png|400px]]
+Figure 2: mRFP1-TEVcs-SRRA reporter gene in its pBbB8k-GFP backbone.
+Fluorescence was quantified after arabinose induction at 1% by reading in a Plate Reader overnight. Here, we measurated the fluorescence of mRFP-TEVcs-SSRA and mRFP-TEVcs.
+[[File:resultK3147003.png|400px]]
+Figure 3 :Measurement of the fluorescence at 30°C and 37°C in RFU of bacteria expressing mRFP-TEVcs or mRFP-TEVcs-SSRA