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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE 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
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 377
  Illegal BglII site found at 2211
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE 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).

Figure 1 Photos of transformants of mec detective system on LB agar broth.

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.

Figure 2 A. Results of the RFP fluorescence intensity and OD600 under different concentrations of Ampicillin. B. Photos of E. Coli DH5α after culturing overnight under 0 and 5 μg/mL Ampicillin..

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.

Figure 3 Curve of RFP fluorescence intensity and culture time under 5 μg/mL Ampicillin, Cephalothin, and Cefoxitin.