Part:BBa_K3147003

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]