Part:BBa_K2150013

A fusion protein combining tetX(BBa_K2150101) with GFP(BBa_E0040)

This protein(refered to as tetX-GFP) combines a tetracycline-degrading enzyme (tetX monooxygenase, BBa_K2150101) and a green fluorescent protein (GFPmt3, BBa_E0040) with a 3*GGGGS linker.

Usage and Biology

TetX-GFP has activities of both tetX and GFP. As a degradation enzyme, it can degrade tetracycline(tc) and its analogs (see BBa_K2150101), and can give the tetracycline resistance to E.coli. As a green fluorescent protein, it inherits the normal function of the GFP. However, we noticed that when expressed under the same promoter, the fluorescence intensity(FI) of tetX-GFP was weaker than that of GFP at the same growth stage of E.coli. The reason for this may be that the expression level of tetX-GFP is lower than GFP under the same promoter or that tetX-GFP has different excitation spectrum and emission spectrum comparing to GFP.

TetX-GFP can accurately reflect the expression level of tetracycline-degrading enzyeme and report whether our system is working (whether our bacteria is degrading tetracycline). When tetX-GFP is expressed under a tetracycline-regulated promoter such as BBa_R0040, it will become a visible tetracycline sensor in the presence of tetR (BBa_C0040), with no need for additional tetracycline-resistance genes.

Characterization

Role As a Degrading enzyme

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 AgeI site found at 562
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1859