Difference between revisions of "Part:BBa K3093008"
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===Characterization===
 
===Characterization===
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In order to characterize cenA-hlyA-hlyB-hlyD, ECUST_iGEMer inserted these genes after a modified pET28b--pIN2, a λ-driven expression and Kan-antibiotic vector .
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In order to characterize cenA-hlyA-hlyB-hlyD, ECUST_iGEMer inserted these genes after a modified pET28b--pIN2, a λ-driven expression and Kan-antibiotic vector.
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α-hemolysin specific signal peptide HlyAs, which corresponds to residues 965-1024 of α-hemolysin, was fused to the C-terminus of cenA and cex by overlapping PCR. In addition, the genes encoding the components of translocator, hlyB and hlyD, were amplified together by PCR using part:BBa_K1166002 as template, cenA was amplified by PCR using part:BBa_K523015. Using seamless cloning we linkers thes three fragments together,it was then transformed into E. coli strain DH5α and positive clones were selected by Kan antibiotics.  
  
 
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<div class="exper-com-box"><img style="width:500px;" src="https://2019.igem.org/File:T--ECUST_China--CenA.png"> <br><span style="font-size: 14px;">  
 
<div class="exper-com-box"><img style="width:500px;" src="https://2019.igem.org/File:T--ECUST_China--CenA.png"> <br><span style="font-size: 14px;">  
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  <b>Figure 1.</b> Gene circuit of cenA-hlyA/hlyBhlyD/pIN2</span><br><span style="font-size: 12px;"><i>cex</i>: Cellulase exoglucanase gene, <i>cenA</i>: Cellulase endoglucanase gene,<i>hlyA, hlyB, hlyD</i>: α-hemolysin system gene</span></div></html>  
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  <b>Figure 1.</b> Gene circuit of cenA-hlyA/hlyBhlyD/pIN2</span><br><span style="font-size: 12px;"><i>cex</i>: Cellulase exoglucanase gene, <i>cenA</i>: Cellulase endoglucanase gene,<i>hlyA, hlyB, hlyD</i>: α-hemolysin system gene</span></div></html>
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===References===
 
===References===

Revision as of 10:29, 21 October 2019


cenA-hlyA+secretion system

The composite part consists of cenA-hlyA-hlyB-hlyD, which expresses C. fimi endoglucanase CenA fused with HlyA, through α-hemolysin secretion system from uropathogenic E. coli, it can be secreted out of E. coli detected in the medium.

Usage and Biology

The secretory CenA consists of CenA (endoglucanase)and α-hemolysin secretion system. All of the basic parts can be found in iGEM standard biological parts, but ECUST_iGEMers combined these basic parts to constitute a new composite part.

By this part we can achieve secreting CenA out of E. coli, but the secretory efficiency was relatively low, and CenA expressed in E. coli has shown limited enzyme activity by our CMC-Na assay and literature results. Both reasons combined contribute to our expected but less satisfying results.

Characterization

In order to characterize cenA-hlyA-hlyB-hlyD, ECUST_iGEMer inserted these genes after a modified pET28b--pIN2, a λ-driven expression and Kan-antibiotic vector. α-hemolysin specific signal peptide HlyAs, which corresponds to residues 965-1024 of α-hemolysin, was fused to the C-terminus of cenA and cex by overlapping PCR. In addition, the genes encoding the components of translocator, hlyB and hlyD, were amplified together by PCR using part:BBa_K1166002 as template, cenA was amplified by PCR using part:BBa_K523015. Using seamless cloning we linkers thes three fragments together,it was then transformed into E. coli strain DH5α and positive clones were selected by Kan antibiotics.


Figure 1. Gene circuit of cenA-hlyA/hlyBhlyD/pIN2
cex: Cellulase exoglucanase gene, cenA: Cellulase endoglucanase gene,hlyA, hlyB, hlyD: α-hemolysin system gene

References

[1]Yokobayashi Y, Weiss R, Arnold FH. Directed evolution of a genetic circuit. Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16587-91. Epub 2002 Nov 25. PubMed PMID: 12451174; PubMed Central PMCID: PMC139187.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1229
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 3156
    Illegal AgeI site found at 3268
  • 1000
    COMPATIBLE WITH RFC[1000]




Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 114
    Illegal NotI site found at 1258
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1171
    Illegal BamHI site found at 62
    Illegal BamHI site found at 307
    Illegal XhoI site found at 71
    Illegal XhoI site found at 669
    Illegal XhoI site found at 918
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 449
    Illegal NgoMIV site found at 1374
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 353