Difference between revisions of "Part:BBa K5101005"

 
 
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<partinfo>BBa_K5101005 short</partinfo>
 
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temperature-inducible suicide module
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__TOC__
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==Usage and Biology==
 
To enable the engineered bacteria to suicide when released into the environment (generally above 39 degrees Celsius) during fecal disposal, we chose a temperature-responsive protein TlpA39(<partinfo>BBa_K2572000</partinfo>) and its corresponding promoter p<i>TlpA</i>(<partinfo>BBa_K2500003</partinfo>) as regulatory elements to induce the expression of the lysis protein PhiX174E. TlpA39 is derived from <i>Salmonella enterica</i>. It is a temperature-sensitive protein that can bind to p<i>TlpA</i>. At temperatures above 39 degrees Celsius, the TlpA39 protein will remain in a monomeric form, thereby losing the ability to bind to the promoter p<i>TlpA</i>. If the temperature drops below 39 degrees Celsius, the TlpA39 protein will form a dimer; the dimer can inhibit the p<i>TlpA</i> and prevent the recruitment of RNA polymerase, thereby suppressing downstream gene expression.
 
To enable the engineered bacteria to suicide when released into the environment (generally above 39 degrees Celsius) during fecal disposal, we chose a temperature-responsive protein TlpA39(<partinfo>BBa_K2572000</partinfo>) and its corresponding promoter p<i>TlpA</i>(<partinfo>BBa_K2500003</partinfo>) as regulatory elements to induce the expression of the lysis protein PhiX174E. TlpA39 is derived from <i>Salmonella enterica</i>. It is a temperature-sensitive protein that can bind to p<i>TlpA</i>. At temperatures above 39 degrees Celsius, the TlpA39 protein will remain in a monomeric form, thereby losing the ability to bind to the promoter p<i>TlpA</i>. If the temperature drops below 39 degrees Celsius, the TlpA39 protein will form a dimer; the dimer can inhibit the p<i>TlpA</i> and prevent the recruitment of RNA polymerase, thereby suppressing downstream gene expression.
  
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==Design==
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===Usage and Biology===
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As shown in Figure 1, we designed the plasmid pET29(a)-p<i>J23119</i>-TIpA39-T-p<i>TIpA</i>-RBS-PhiX174E-T7 using SnapGene software. Through the continuous expression of the TIpA39 protein under the strong constitutive promoter <i>J23119</i>, the p<i>TIpA</i> promoter is inhibited at temperatures below 39 degrees Celsius. When animal manure is being processed and the temperature reaches above 39 degrees Celsius, it will initiate the expression of the lysis gene, achieving the self-destructive effect. In the future, we will complete wet experimental validation of this suicide module.
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<img src="https://static.igem.wiki/teams/5101/partpage/pet29-a-pj23119-tlpa39-t-ptlpa-rbs-phix174e-t7-map.png" alt="plasmid1" width="300">
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<p align="center"><b>Figure 1</b>  Plasmid pET29(a)-p<i>J23119</i>-TIpA39-T-p<i>TIpA</i>-RBS-PhiX174E-T7</p>
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==Sequence and Features==
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K5101005 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5101005 SequenceAndFeatures</partinfo>
  

Latest revision as of 13:16, 11 September 2024


pET29(a)-pJ23119-TIpA39-T-pTIpA-RBS-PhiX174E-T7


temperature-inducible suicide module

Usage and Biology

To enable the engineered bacteria to suicide when released into the environment (generally above 39 degrees Celsius) during fecal disposal, we chose a temperature-responsive protein TlpA39(BBa_K2572000) and its corresponding promoter pTlpA(BBa_K2500003) as regulatory elements to induce the expression of the lysis protein PhiX174E. TlpA39 is derived from Salmonella enterica. It is a temperature-sensitive protein that can bind to pTlpA. At temperatures above 39 degrees Celsius, the TlpA39 protein will remain in a monomeric form, thereby losing the ability to bind to the promoter pTlpA. If the temperature drops below 39 degrees Celsius, the TlpA39 protein will form a dimer; the dimer can inhibit the pTlpA and prevent the recruitment of RNA polymerase, thereby suppressing downstream gene expression.

Design

As shown in Figure 1, we designed the plasmid pET29(a)-pJ23119-TIpA39-T-pTIpA-RBS-PhiX174E-T7 using SnapGene software. Through the continuous expression of the TIpA39 protein under the strong constitutive promoter J23119, the pTIpA promoter is inhibited at temperatures below 39 degrees Celsius. When animal manure is being processed and the temperature reaches above 39 degrees Celsius, it will initiate the expression of the lysis gene, achieving the self-destructive effect. In the future, we will complete wet experimental validation of this suicide module.

plasmid1

Figure 1 Plasmid pET29(a)-pJ23119-TIpA39-T-pTIpA-RBS-PhiX174E-T7

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 1205
    Illegal NotI site found at 572
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 857
    Illegal XhoI site found at 1632
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1160
  • 1000
    COMPATIBLE WITH RFC[1000]