Part:BBa_K1639023

GFP with an ASV degradation tag with a half-life of ~110 min

GFP is seemed to be very stable once produced. But ASV tagged GFP is susceptible to action of intracellular proteases which reduce it's half-life approximately to 110 min. ASV tag consist of C-terminal extension sequences RPAANDENYAASV It's useful for studying of real-time gene expression in individual cells.

Usage and Biology

To expand the use of GFP as a reporter protein, new variants have been constructed by the addition of short peptide sequences to the C-terminal end of intact GFP. This rendered the GFP susceptible to the action of indigenous housekeeping proteases, resulting in protein variants with half-lives ranging from 40 min to a few hours when synthesized in Escherichia coli and Pseudomonas putida. In our project the ASV tagged variant with 110 min half-life.

(A) Stability of Gfp variants in E. coli following a downshift. •, MV1190(λ-pir)(pJBA28) expressing Gfpmut3*; ×, MV1190(λ-pir)(pJBA120) expressing Gfp(ASV); ▪, MV1190(λ-pir)(pJBA118) expressing Gfp(AAV); ▴, MV1190(λ-pir)(pJBA116) expressing Gfp(LVA); and ⧫, MV1190(λ-pir)(pJBA114) expressing Gfp(LAA). (B) Stability of Gfp variants in P. putida following a downshift. •, KT2442 (lacIq) expressing Gfpmut3*; ×, KT2442 (lacIq) expressing Gfp(ASV); ▪, KT2442 (lacIq) expressing Gfp(AAV); ▴, KT2442 (lacIq) expressing Gfp(LVA); and ⧫, KT2442 (lacIq) expressing Gfp(LAA).

A major drawback of GFP is that once formed it seems to be very stable, which in turn renders the protein less valuable for studies of transient (real-time) gene expression. The slope constant, μ, were determined to be approximately −0.0062 min−1. This corresponds to in vivo half-life of mature Gfp(ASV) (T1/2 = −ln 2/μ) 110 min.

Microscopic images of ASV tagged GFP As shown in figure the ASV tag(RPAANDENYAASV) doesn't affect flourscence of GFP

References

Andersen JB, Sternberg C, Poulsen LK, Bjørn SP, Givskov M, Molin S. New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria. Applied and Environmental Microbiology. 1998;64(6):2240-2246. 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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 644