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Part:BBa_K3152018
mec detective system
It is the key part that is responsible for β-Lactams report. It contains 4 parts: mecRI, mecI, mec operator and mCherry. A certain content of β-lactams will change the structure of mecR1 so that it could degrade mecI repressor and release mec opeator. As a result, the mec operator promote the mCherry gene and cause mCherry to glom.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1790
Illegal NheI site found at 1813
Illegal NheI site found at 1839
Illegal NheI site found at 1862 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 377
Illegal BglII site found at 2211 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1456
Usage and Biology
We took out two colonies from the LB solid medium and digested by EcoRI and PstI restriction enzymes to further verify the correctness of the connection (Figure 1).
Then we tested the effect when β- lactam antibiotics existed. Ampicillin were added into 0.8 mL LB broth with a gradient final concentrations, samples were collected after culturing in 37 ℃ overnight. As shown in Figure 2A, we can obviously see the intensity of red fluorescence increased with the concentration of ampicilin going up before reaching inhibited concentration. The best reaction concentration is 5 μg/mL, which show the most obvious report. The consequences of Figure 2B also support the results.
To further improve the accuracy of the Mec detection system towards β-lactam antibiotics, we tried with three different β-lactam antibiotics: ampicillin, cephalothin, cefoxitin. The culture without antibiotics was used as a control group. Figure 3 showed that with the extension of culture time, the fluorescence of mCherry in test groups gradually increased, which indicates that the expression level of mCherry in the medium gradually increased. This results revealed that the sensitive of our Mec detection system is best for ampicillin.
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