Difference between revisions of "Part:BBa K3147003"

Latest revision as of 07:32, 21 October 2019

mRFP1 fused to a TEV-cleavable ssrA tag

I : parts BBa_K3147003 function

This construction produces an mRFP1 Part:BBa_E1010[1] fused in C-ter with a ssrA fast degradation tag [2] Part:BBa_M0050. The TEV cutting site (Part:BBa_J18918) was added between the mRFP1 and the ssrA tag. This construction was used to test the specificity of KARMA using a VHH targeting GFP. In the presence of TEV the ssrA is cleaved and mRFP1 is not degraded anymore.

Figure 1: Construct Design: mRFP1 fused to an ssrA proteolysis tag with a TEV cutting site between the two.

II. Proof of function

This construction was cloned by Gibson Assembly in a pBbB8k-GFP (https://www.addgene.org/35363/ ) backbone under the control of a pBAD promoter.

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

We compared the basal fluorescence of strain E. coli NEB10β transformed with the mRFP1-TEVcs construction to an E. coli NEB10β strain transformed with the mRFP1-TEVcs-ssrA construction. Fluorescence was quantified after overnight induction with 1% arabinose on a plate reader.

Below are the fluorescence measurements of the mRFP1-TEVcs-ssrA and of the mRFP1-TEVcss at 30 and 37°C.

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

We also did statistical analysis of the fluorescence from both construct with a Welch two sample test by normalizing the data,taking the version without proteolysis tag as 100%.

Figure 4: Statistical analysis of the effect of ssrA tag on the mRFP-TEVcs at 30°C

At 30°C, the ssrA tag reduce by 99% the fluorescence obtained by mRFP-TEVcs. The p-value obtained is 0.0006.

Figure 5: Statistical analysis of the effect of ssrA tag on the mRFP-TEVcs at 37°C

At 37°C, the ssrA tag reduce by 96% the fluorescence obtained by mRFP-TEVcs. The p-value calculated is 6.5e-07.

Reference

[1] Levraud, Jean-Pierre et al. 2007. « Identification of the Zebrafish IFN Receptor: Implications for the Origin of the Vertebrate IFN System ». The Journal of Immunology 178(7): 4385‑94. doi:10.4049/jimmunol.178.7.4385

[2] 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
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]

@@ Line 1: / Line 1: @@
 __NOTOC__
-<partinfo>BBa_K3147003 short</partinfo>
+<div align="center"><partinfo>BBa_K3147003 short</partinfo></div>
+===I : parts BBa_K3147003 function===
+This construction produces an mRFP1 [[Part:BBa_E1010]][1] fused in C-ter with a ssrA fast degradation tag  [2] [[Part:BBa_M0050]]. The TEV cutting site ([[Part:BBa_J18918]]) was added between the mRFP1 and the ssrA tag. This construction was used to test the specificity of KARMA using a VHH targeting GFP. In the presence of TEV the ssrA is cleaved and mRFP1 is not degraded anymore.
-===I : parts BBa_K3147003 (mRFP1-TEVcs-SSRA) function===
-The Montpellier 2019 team made a reporter gene construction in order to carry out their proof of concept. This construction produces an mRFP1 (BBA_E1010)[1] fused in C-ter with a fast degradation tag called ssrA  [2] (BBA_M0050). The TEV cutting site (BBa_J18918) was added between the mRFP1 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 mRFP to glow without being degraded.
   <div align="center">[[File:designK3147003.png|650px]]</div>
-  <div align="center"><b>Figure 1</b>: Construct Design: mRFP1 fused to an SSRA proteolysis tag with a TEV cutting site between the two.</div>
+  <div align="center"><b>Figure 1</b>: Construct Design: mRFP1 fused to an ssrA proteolysis tag with a TEV cutting site between the two.</div>
 ===II. Proof of function===
-The experimental approach to test the activity of these reporters was to compare the basal fluorescence rate of mRFP1 with a  TEV cutting site "cleaved" to this construction. For this purpose we made a control construction: mRFP1-TEVcs (BBa_K3147004) similar to this one by removing the proteolysis tag and simulating a cut by the TEV. The construction was cloned by Gibson Assembly in a pBbB8k-GFP (https://www.addgene.org/35363/ ) backbone under the control of a BAD type promoter, in order to control its expression.
+This construction was cloned by Gibson Assembly in a pBbB8k-GFP (https://www.addgene.org/35363/ ) backbone under the control of a pBAD promoter.
   <div align="center">[[File:PlasmideK3147003.png|400px]]</div>
   <div align="center"><b>Figure 2</b>: mRFP1-TEVcs-ssrA reporter gene in its pBbB8k-GFP backbone.</div>
-We compared the basal fluorescence of strain E. coli NEB10β transformed with the mRFP1-TEVcs construction to an E. coli NEB10β strain transformed with the mRFP1-TEVcs-ssrA construction. Fluorescence was quantified after induction with arabinose concentrated at 1% with the plate reader overnight. Here are the fluorescence of mRFP-TEVcs-ssrA and mRFP-TEVcs at 30°C and 37°C. We can see that the ssrA tag is reducing the fluorescence from the mRFP-TEVcs.
+We compared the basal fluorescence of strain <i>E. coli</i> NEB10β transformed with the mRFP1-TEVcs construction to an <i>E. coli</i> NEB10β strain transformed with the mRFP1-TEVcs-ssrA construction. Fluorescence was quantified after overnight induction with 1% arabinose on a plate reader.
+Below are the fluorescence measurements of the mRFP1-TEVcs-ssrA and of the mRFP1-TEVcss at 30 and 37°C.
   <div align="center">[[File:resultK3147003.png|500px]]</div>
-  <div align="center"><b>Figure 3</b>: Measurement of the fluorescence at 30°C and 37°C of bacteria expressing mRFP-TEVcs or mRFP-TEVcs-ssrA in RFU</div>
+  <div align="center"><b>Figure 3</b>: Measurement of the fluorescence at 30°C and 37°C of bacteria expressing mRFP-TEVcs or mRFP-TEVcs-ssrA</div>
+We also did statistical analysis of the fluorescence from both construct with a Welch two sample test by normalizing the data,taking the version without proteolysis tag as 100%.
+<div align="center">[[File:Stats30CK3147003.png|500px]]</div>
+ <div align="center"><b>Figure 4</b>: Statistical analysis of the effect of ssrA tag on the mRFP-TEVcs at 30°C</div>
+At 30°C, the ssrA tag reduce by 99% the fluorescence obtained by mRFP-TEVcs. The p-value obtained is 0.0006.
+<div align="center">[[File:Stats37CK3147003.png|500px]]</div>
+ <div align="center"><b>Figure 5</b>: Statistical analysis of the effect of ssrA tag on the mRFP-TEVcs at 37°C</div>
+At 37°C, the ssrA tag reduce by 96% the fluorescence obtained by mRFP-TEVcs. The p-value calculated is 6.5e-07.
 ==Reference==