Part:BBa_K3147000

sfGFP fuse to TEV-cleavable ssrA tag

I : parts BBa_K3147000 function

The Montpellier 2019 team submitted a reporter gene construction in order to carry out their proof of concept. This construction produces an sfGFP(bs) [1] [2] [3] Part:BBa_K1365020 fused in C-terminal with a fast degradation tag called ssrA [4] [5] Part:BBa_M0050. The TEV cutting site Part:BBa_J18918 has been added between the sfGFP and the ssrA tag. This construction can be used as a reporter for proteolysis activity by TEV. In the presence of TEV the ssrA is cleaved and sfGFP is not degraded anymore.

Figure 1 : Construct Design: sfGFP fused to an ssrA proteolysis tag with a TEV cutting site in between.

II. Proof of function

As a positive control, we built a construct simulating a ssrA cleavage by the TEV, by expressing sfGFP with a C-terminal sequence corresponding to the cleaved TEV recognition Part:BBa_K3147001. We expressed this part in a plasmid under the control of the arabinose promoter: pBbE8K-RFP backbone (https://www.addgene.org/35327/). We cloned it by Gibson Assembly.

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

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 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. We can see that the ssrA system is more efficient at 37C as described in part M0050 characterization.

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.

[4] Fernandez-Rodriguez, Jesus, et Christopher A. Voigt. 2016. « Post-TranslationalControl of Genetic Circuits Using Potyvirus Proteases ». Nucleic Acids Research 44(13): 6493‑6502.

[5] 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
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 41
  Illegal NheI site found at 53
  Illegal NheI site found at 76
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 82
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]