Part:BBa_K3378001

ClyR fused with PhoA signal peptide

ClyR is a chimeric lysine which can bind to S. mutans by a cell-wall binding domain and then lysis its cell wall from outside by a catalytic domain. ClyR has no activity for Gram-negative bacteria and can be expressed in Escherichia coli. PhoA fused ClyR can be secreted to the outside of the cells, killing S. mutans in the environment.

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 826
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 190
  Illegal AgeI site found at 274
  Illegal AgeI site found at 589
  Illegal AgeI site found at 772
  
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

To demonstrate the secretion of PhoA fused ClyR, plasmid pET-28a(+)-ClyR (with PhoA) was transferred to E. coli BL21(DE3), same plasmid just without PhoA signal peptide as control. E. coli strains was cultured to OD₆₀₀~0.6, induced with 0.2 mM IPTG and 1 % glycine, and then overnight cultured at 16 ℃. On the second day, take the supernatant and bacterial liquid for SDS analysis, thus we can demonstrate that the PhoA fused ClyR can be secreted to the medium effectively (Figure 1).

Figure 1. SDS-PAGE analysis of ClyR secretion. BL-PhoA- Bacteria liquid of PhoA nagtive group; S-PhoA- Supernatant of PhoA negative group; BL-PhoA+ Bacteria liquid of PhoA positive group; S-PhoA+ Supernatant of PhoA positive group;

Reference

[1] Yang, Hang, et al. "A chimeolysin with extended-spectrum streptococcal host range found by an induced lysis-based rapid screening method." Scientific reports 5 (2015): 17257.
[2] Yang, Hang, et al. "Antibiofilm activities of a novel chimeolysin against Streptococcus mutans under physiological and cariogenic conditions." Antimicrobial agents and chemotherapy 60.12 (2016): 7436-7443.
[3] Xu, Jingjing, et al. "Activity of the chimeric lysin ClyR against common Gram-positive oral microbes and its anticaries efficacy in rat models." Viruses 10.7 (2018): 380.