Part:BBa_K3606108

P4 driven mcbABCD

BBa_K3606104 is one of a series of composite parts designed to screen a suitable strength promoter for mcbABCD.

Usage and Biology:

P4, which is a constitutive promoter, will continuously drive expression of mcbABCD. McbABCD is responsible for producing MccB17.

Design:

In our design the proper expression level of mcbABCD is very important. Since its product MccB17 acts as antibiotic, its low expression will make it unable to kill the surrounding bacteria and cannot improve its competitiveness in the intestine. But considering its toxicity, its high expression will also increase the burden of survival of engineered bacteria. So we need to test a collection of promoters, and find the most suitable one for it.

Characterization:

In order to prove the efflux and immune effects of McbEFG, we compared E.coli with mcbABCD-mcbEFG-ptetR and E.coli with mcbABCD to observe the difference of their antibacterial effects. We mixed WT E.coli (expressing GFP driven by plac) and E.coli with mcbABCD-mcbEFG-ptetR in different ratios (5:7 20:7 50:7), and measured the OD value of the bacteria two hours after adding an inducer, which can be used to reflect the antibacterial effect of antimicrobial peptides. We followed the same method as above to mix WT E. coli and E.coli with merely mcbABCD, induce and measure the OD value.

Figure1. MEFL/particle for different Px and PxAD when the ratio of mixure(WT:Px=5:7)

Figure2. MEFL/particle for different Px and PxAD when the ratio of mixure(WT:Px=20:7)

Figure3. MEFL/particle for different Px and PxAD when the ratio of mixure(WT:Px=50:7)

The result shows that E.coli with mcbABCD-mcbEFG-ptetR can more effectively inhibit the metabolism of other bacteria than those merely with mcbABCD in each group. With mcbEFG, E.coli are able to export antimicrobial peptides more effectively and supposed to have better immunity themselves so as to affect the surrounding WT’s metabolism(the expression of GFP), and better survive in the environment. While comparing to the control group, we can also see a significant decrease in the GFP expression result when there are E.coli with mcbABCD-mcbEFG-ptetR or mcbABCD, which indicates that the antimicrobial peptide(mccb17) encoded by mcbABCD does have a strong restraining effect on other microbial.

Further Application:

To use this part, simply clone it into a low-copy plasmid vector, transfer into E.coli, incubate overnight. You can also understand the strength of P4 through my experimental results.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 2327
  Illegal PstI site found at 2360
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 2327
  Illegal PstI site found at 2360
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 2327
  Illegal PstI site found at 2360
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 2327
  Illegal PstI site found at 2360
  Illegal NgoMIV site found at 1989
  Illegal AgeI site found at 2161
  
• 1000
  COMPATIBLE WITH RFC[1000]