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
The E.coli constitutively expressing tetX-GFP (expressed from ptet,BBa_R0040) is able to survive on the LB agar plate with high concentration of tetracycline, while the wild type (DH5alpha) and the E.coli expressing only GFP is not. [[File:tetX-GFP_1.png|200px|thumb|left|alt text]
The E.coli constitutively expressing tetX-GFP has the ability to degrade tetracycline. After 6-hour incubation in M9 containing 100uM tetracycline, the tetracycline whose concentration is represented by the absorbance at 360nm was degraded by about 15%.
Role as a green fluorescent enzyme
The FI of E.coli constitutively expressing tetX-GFP is increasing as the cell growth. However, at the same OD600, the FI of the tetX-GFP group is weaker than that of GFP group.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 562
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1859
| None |