Difference between revisions of "Part:BBa K4645020"

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<partinfo>BBa_K4645020 short</partinfo>
 
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CSP is a quorum sensing signaling molecule of <i>S. mutans</i>, and its concentration is positively correlated with the amount of <i>S. mutans</i>. 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 <i>S. mutans</i> and avoids the biofilm formed by <i>S. mutans</i> on the surface of our bacteria.
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CSP is a quorum sensing signaling molecule of <i>S. mutans</i>, and its concentration is positively correlated with the amount of <i>S. mutans</i>. 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 <i>nlmC</i> 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 <i>S. mutans</i> and avoids the biofilm formed by <i>S. mutans</i> on the surface of our bacteria.
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<p>The design circuit of the adhesion part is as follows (<b>Figure 1</b>).</p>  
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<p>The design circuit of the adhesion part is as follows.</p>  
 
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<img src=https://static.igem.wiki/teams/4645/wiki/project/design/sterilization-circuit.png width=100% alt="">
 
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<center><b>Figure 1.</b> Gene circuit of adhesion part.</center>
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<center><b>Figure 1.</b> The outline of genetic circuit.</center>
 
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===Methods and Result===
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<p><b>Verification of bactericidal activity</b></p>
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<p>To determine the dose dependence and time dependence of ClyR's lytic activity, <i>S. mutans</i> UA159 was resuspended to an optical density (OD<sub>600</sub>) of ~0.8 using PBS buffer. In a 96-well plate, we sequentially added 35 μL of ClyR solution at different concentrations and 165 μl of bacterial suspension. The OD<sub>600</sub> was continuously measured using a <b>Synergy</b><sup>TM</sup> H1 microplate reader under constant shaking at 37℃ for 1 hour. The experimental results are shown in the figure below.
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<img src=https://static.igem.wiki/teams/4645/wiki/wet-lab/clyr/clyr2.jpg width=100% alt="">
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<center><b>Figure 2.</b> Variation curve of OD<sub>600</sub> of <i>S.mutans</i> UA159 treated with different ClyR concentrations. </center></center>
 
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Latest revision as of 15:06, 12 October 2023


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. 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 35 μL of ClyR solution at different concentrations and 165 μl of bacterial suspension. The OD600 was continuously measured using a SynergyTM 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.