Difference between revisions of "Part:BBa K2150013"
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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(Fig.1). | 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(Fig.1). | ||
| − | [[File:tetX-GFP_1.png|250px | + | [[File:tetX-GFP_1.png|250px]] |
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% (Fig.2). | 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% (Fig.2). | ||
Revision as of 19:18, 19 October 2016
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(Fig.1).
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% (Fig.2).
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 (Fig.3).
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