Difference between revisions of "Part:BBa K2922009"
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<partinfo>BBa_K2922009 short</partinfo>
 
<partinfo>BBa_K2922009 short</partinfo>
  
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Kil protein accumulates in the periplasmic space of the bacteria, the periplasmic space was increased and the membrane permeability of bacteria was improved, thereby the protein inside the bacteria can secrete out of bacteria. But only when the intensity of the promoter is proper, the secretion can be achieved and prevent cell lysis and death. Here we use different constitutive promoters to regulate the expression of kil protein to enhance its secretion ability.
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Kil protein (<partinfo>BBa_K1350001</partinfo>)accumulates in the periplasmic space of the bacteria, the periplasmic space was increased and the membrane permeability of bacteria was improved, thereby the protein inside the bacteria can secrete out of bacteria. But only when the intensity of the promoter is proper, the secretion can be achieved and prevent cell lysis and death. Here we use different constitutive promoters to regulate the expression of kil protein to enhance its secretion ability.<ref>http://2014.igem.org/Team:SZU-China/Project/Kil</ref>
  
  
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===Biology===
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===Biology and Usage===
 
In wild-type ''E.coli'' exist a plasmid named colE1, the kil gene of the ColE1 plasmid encodes a peptide that, at low levels, causes the release of periplasmic proteins without cell lysis. In contrast, high-level induction results in cell lysis and death. This indicates that the regulation of kil gene expression is critical for utilization in a protein secretion system.
 
In wild-type ''E.coli'' exist a plasmid named colE1, the kil gene of the ColE1 plasmid encodes a peptide that, at low levels, causes the release of periplasmic proteins without cell lysis. In contrast, high-level induction results in cell lysis and death. This indicates that the regulation of kil gene expression is critical for utilization in a protein secretion system.
  
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The main problem when using constitutive promoters for kil gene expression is the rapid decrease of the viability of bacterial cells before a sufficient amount of target protein has been produced. Using the kil gene under the control of the weak constitutive promoter enabled viability to be maintained.<ref>G. Miksch, E. Fiedler, P. Dobrowolski, K. J. A. o. M. Friehs, The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase. 167, 143-150 (1997).</ref>
 
The main problem when using constitutive promoters for kil gene expression is the rapid decrease of the viability of bacterial cells before a sufficient amount of target protein has been produced. Using the kil gene under the control of the weak constitutive promoter enabled viability to be maintained.<ref>G. Miksch, E. Fiedler, P. Dobrowolski, K. J. A. o. M. Friehs, The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase. 167, 143-150 (1997).</ref>
  
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Here, we use <partinfo>BBa_J23109</partinfo>, <partinfo>BBa_J23112</partinfo> and <partinfo>BBa_J23109</partinfo> to demonstrate the effect of the kil gene controlled by the J21309 series promoters (<partinfo>BBa_J23109</partinfo>) on the release of periplasmic enzymes into the extracellular medium. We fused a synthetic DNA region containing the promoter of the J23109/J23112/J23114 genes with the kil gene and constructed secretion cassettes, where target genes <partinfo>BBa_K118022</partinfo> of interest can be easily integrated.  
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Here, we use <partinfo>BBa_J23109</partinfo>, <partinfo>BBa_J23112</partinfo> and <partinfo>BBa_J23109</partinfo> to demonstrate the effect of the kil gene controlled by the J21309 series promoters (<partinfo>BBa_J23109</partinfo>) on the release of periplasmic enzymes into the extracellular medium. We fused a synthetic DNA region containing the promoter of the J23109/J23112/J23114 genes with the kil gene and constructed secretion cassettes, where target genes-''cex'' <partinfo>BBa_K118022</partinfo> of interest can be easily integrated.  
  
 
<html>
 
<html>
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</html>
 
</html>
  
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===Usage===
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Target genes-''cex'' was inserted into the expression vectors with T7 and RBS (<partinfo>BBa_K525998</partinfo>), then constructed the final parts(<partinfo>BBa_K2922018</partinfo>, <partinfo>BBa_K2922019</partinfo> and <partinfo>BBa_K2922020</partinfo>). We transformed the constructed plasmid into ''E. coli'' BL21 (DE3). After confirmed by the same method, the positive clones were cultivated and induced by IPTG.
  
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In the meantime, we also cultivated the strain with ''T7-RBS-yebF-cex'' (<partinfo>BBa_K1180119</partinfo>) to compare YebF secretion system with Kil secretion system.
  
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===Characterization===
 
  
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===Characterization==
  
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'''1. SDS-PAGE'''
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'''MUC Test'''
  
  
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'''2. HPLC'''
 
  
  
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===Reference===
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Reference
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<references/>
 
<references/>
  

Revision as of 17:45, 19 October 2019


Constitutive promoter J23109, strong RBS and Kil protein combination for high secretion levels

Kil protein (BBa_K1350001)accumulates in the periplasmic space of the bacteria, the periplasmic space was increased and the membrane permeability of bacteria was improved, thereby the protein inside the bacteria can secrete out of bacteria. But only when the intensity of the promoter is proper, the secretion can be achieved and prevent cell lysis and death. Here we use different constitutive promoters to regulate the expression of kil protein to enhance its secretion ability.[1]


Biology and Usage

In wild-type E.coli exist a plasmid named colE1, the kil gene of the ColE1 plasmid encodes a peptide that, at low levels, causes the release of periplasmic proteins without cell lysis. In contrast, high-level induction results in cell lysis and death. This indicates that the regulation of kil gene expression is critical for utilization in a protein secretion system.


The main problem when using constitutive promoters for kil gene expression is the rapid decrease of the viability of bacterial cells before a sufficient amount of target protein has been produced. Using the kil gene under the control of the weak constitutive promoter enabled viability to be maintained.[2]

Here, we use BBa_J23109, BBa_J23112 and BBa_J23109 to demonstrate the effect of the kil gene controlled by the J21309 series promoters (BBa_J23109) on the release of periplasmic enzymes into the extracellular medium. We fused a synthetic DNA region containing the promoter of the J23109/J23112/J23114 genes with the kil gene and constructed secretion cassettes, where target genes-cex BBa_K118022 of interest can be easily integrated.


Target genes-cex was inserted into the expression vectors with T7 and RBS (BBa_K525998), then constructed the final parts(BBa_K2922018, BBa_K2922019 and BBa_K2922020). We transformed the constructed plasmid into E. coli BL21 (DE3). After confirmed by the same method, the positive clones were cultivated and induced by IPTG.

In the meantime, we also cultivated the strain with T7-RBS-yebF-cex (No part name specified with partinfo tag.) to compare YebF secretion system with Kil secretion system.


=Characterization

MUC Test




Reference

  1. ↑ http://2014.igem.org/Team:SZU-China/Project/Kil
  2. ↑ G. Miksch, E. Fiedler, P. Dobrowolski, K. J. A. o. M. Friehs, The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase. 167, 143-150 (1997).

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
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]