Part:BBa_K3093008
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1229
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 3156
Illegal AgeI site found at 3268 - 1000COMPATIBLE WITH RFC[1000]
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 114
Illegal NotI site found at 1258 - 21INCOMPATIBLE 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 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 449
Illegal NgoMIV site found at 1374 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 353
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