Part:BBa_K3606065

P9 driven mcbABCD and PtetR driven mcbEFG

We used McbA-D to inhibit the growth of other bacteria in human intestine so as to enhance Nissle's competitiveness,as well as to reduce the risk of illness caused by some opportunistic pathogens. We introduced McbE-G to inhibit the growth of other bacteria in human intestine so as to enhance Nissle's competitiveness,as well as to reduce risk of illness caused by some opportunistic pathogens.At the same time, we put McbE-G under the control of pTetR in order to response to AHL-LuxR.

Design:

Here, we tried to improve the former antimicrobial peptide(mccb17) expressing system of 2019 Fudan BBa_K3245010. By dividing into the peptide expressing parts and the immunity parts, we wanted to manipulate their expression level with more efficiency.

Figure. mcbABCD Figure. mcbEFG

Usage and Biology:

We introduced mcbABCD to our improved antibiotic expressing system to inhibit the growth of other bacteria in human intestine so as to enhance Nissle's competitiveness, as well as to reduce risk of illness caused by some opportunistic pathogens.Driven by a series of constitutive promoters with different strength which include P9, mcbABCD is expressed in E. coli in a controlled, reliable manner.

McbEFG works as an adjusting part in quorum sensing system. When the number of engineered bacteria is low, the part will help export antibiotic and provide self immunity in order to help proliferate. PtetR determines the expression of mcbEFG when the number of bacteria is at different level.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 5547
  Illegal XbaI site found at 5532
  Illegal PstI site found at 2370
  Illegal PstI site found at 2403
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 5547
  Illegal PstI site found at 2370
  Illegal PstI site found at 2403
  Illegal NotI site found at 5539
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 5547
  Illegal BamHI site found at 3366
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 5547
  Illegal XbaI site found at 5532
  Illegal PstI site found at 2370
  Illegal PstI site found at 2403
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 5547
  Illegal XbaI site found at 5532
  Illegal PstI site found at 2370
  Illegal PstI site found at 2403
  Illegal NgoMIV site found at 2032
  Illegal AgeI site found at 2204
  
• 1000
  COMPATIBLE WITH RFC[1000]

Results:

We constructed the plasmid and successfully expressed the system, here is the electrophoresis map. 图

Further Application:

For futher application, this part expresses antimicrobial peptide(mccb17) as well as providing immunity to create survival advantage for the engineered strain. These part are especially useful to be expressed in vivo because research has proved that it can also ease the inflammation in intestine by limiting the expansion of related pathogens and pathobionts.

References:

Collin F, Maxwell A. The Microbial Toxin Microcin B17: Prospects for the Development of New Antibacterial Agents. J Mol Biol. 2019;431(18):3400–3426. doi:10.1016/j.jmb.2019.05.050

S. Duquesne, D. Destoumieux-Garzón, J. Peduzzi, S. Rebuffat. Microcins, gene-encoded antibacterial peptides from enterobacteria

Sassone-Corsi M, Nuccio SP, Liu H, Hernandez D, Vu CT, Takahashi AA, Edwards RA, Raffatellu M. Microcins mediate competition among Enterobacteriaceae in the inflamed gut. Nature. 2016 Dec 8;540(7632):280-283.

Mutalik VK, Guimaraes JC, Cambray G, Lam C, Christoffersen MJ, Mai QA, Tran AB, Paull M, Keasling JD, Arkin AP, Endy D. Precise and reliable gene expression via standard transcription and translation initiation elements. Nat Methods. 2013 Apr;10(4):354-60. doi: 10.1038/nmeth.2404. Epub 2013 Mar 10. PMID: 23474465.