Part:BBa_K4645020

The two-component system of S. mutans senses the CSP to regulate the expression of ClyR

CSP is a quorum sensing signaling molecule of S. mutans, and its concentration is positively correlated with the amount of S. mutans. ComD is a membrane-bound histidine kinase (HK) capable of autophosphorylation in response to sensing CSP. The response regulatory factor (RR) ComE then catalyzes the transfer of phosphorylated groups to aspartic residues in its receptor region. Phosphorylated ComE (P-ComE) is allosterically transformed into an active conformation that binds upstream to the CSP-sensitive promoter nlmC and activates transcription. Then expressing ClyR, a chimeric lysin, is composed of a catalytic domain from PlyC and a cell-wall binding domain from PlySs2. ClyR can recognize and lyse the cell wall of S. mutans and avoids the biofilm formed by S. mutans on the surface of our bacteria.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 627
  Illegal EcoRI site found at 1985
  Illegal XbaI site found at 3288
  Illegal PstI site found at 1027
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 627
  Illegal EcoRI site found at 1985
  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal PstI site found at 1027
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 627
  Illegal EcoRI site found at 1985
  Illegal XhoI site found at 3240
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 627
  Illegal EcoRI site found at 1985
  Illegal XbaI site found at 3288
  Illegal PstI site found at 1027
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 627
  Illegal EcoRI site found at 1985
  Illegal XbaI site found at 3288
  Illegal PstI site found at 1027
  Illegal AgeI site found at 2608
  Illegal AgeI site found at 2692
  Illegal AgeI site found at 3007
  Illegal AgeI site found at 3190
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1823
  Illegal BsaI.rc site found at 2262
  

Gene Design in Circuits

The design circuit of the adhesion part is as follows (Figure 1).

Figure 1. The outline of genetic circuit.

Methods and Result

Verification of bactericidal activity

To determine the dose dependence and time dependence of ClyR's lytic activity, S. mutans UA159 was resuspended to an optical density (OD₆₀₀) of ~0.8 using PBS buffer. In a 96-well plate, we sequentially added 35μL of ClyR solution at different concentrations and 165μl of bacterial suspension. The OD₆₀₀ was continuously measured using a Synergy H1 microplate reader under constant shaking at 37℃ for 1 hour. The experimental results are shown in the figure below.

Figure 2. Variation curve of OD₆₀₀ of S.mutans UA159 treated with different ClyR concentrations.