Part:BBa_K3147003

mRFP1 fused to a TEV-cleavable ssrA tag

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) 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.

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

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.

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 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.

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

Reference

[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]