Part:BBa_K3606003

mcbABCD

This part produces an antibiotic originated from Escherichia coli that targets a bacterial topoisomerase, DNA gyrase.

Background:

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 firstly test whether the polycistron could work properly and separately, then manipulate their expression level with more efficiency.

Design:

This part is an antibiotic coding gene cluster with leader peptide and proteins for its maturation.

Figure1. structure of mcbABCD

Results

We have successfully expressed mcbA and mcb C for the fundamental validation of the antimicrobial peptide(mccb17) expression. We've constructed the plasmid for mcbB and mcbD expression, yet the results of colony PCR is false positive thus further validation is needed. Figure2. gel map for mcbA, mcbC, mcbE and mcbF

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 2297
  Illegal PstI site found at 2330
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 2297
  Illegal PstI site found at 2330
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 2297
  Illegal PstI site found at 2330
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 2297
  Illegal PstI site found at 2330
  Illegal NgoMIV site found at 1959
  Illegal AgeI site found at 2131
  
• 1000
  COMPATIBLE WITH RFC[1000]

Further Application

For futher application, this part is provided as an antimicrobial peptide(mccb17) expressing example and are effective for a wide range of microbes. This part can be used together with BBa_K3606004 to provide immunity for the engineered strain and create survival advantage. 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.

Reference

[1] 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

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

[3] 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.