Part:BBa_K3093009

cex-HlyA+secretion system

The composite part consists of cex-hlyA-hlyB-hlyD, which expresses C. fimi exoglucanase Cex 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 Cex consists of Cex (β-1,4-exoglucanase) 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 Cex out of E. coli, but the secretory efficiency was relatively low, and Cex 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 cex-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, cex was amplified from the gene synthesized by Genewiz, and the sequence was obtained from ncbi. Using seamless cloning we linkers these 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 cex-hlyA/hlyBhlyD/pIN2
cex: Cellulase exoglucanase gene, cex: Cellulase exoglucanase gene,hlyA, hlyB, hlyD: α-hemolysin system gene