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
Positive clones were further inoculated overnight in 4 mL LB broth with proper antibiotic concentration. The next day, we transferred 1 mL culture to 100mL LB in a 37℃ shaker proper antibiotics added. For approximately 3hrs, O.D. of cell growth was measured, until it reached 0.6, we placed the shaker in 30℃ at 220rpm for induce. After 8 hours, the whole culture medium was spun down at 4000rpm for 10 minutes at 4℃. After that, we further used centrifugation of 1200rpm for 10 minutes at 4℃ to further discard all non-soluble parts of cells and supernatant was collected. Next, we applied a 3kDa protein concentration tube to concentrate the supernatant by 20 times for better visualization. The concentrated solution was used as crude Cex solution.
Due to Cex enzyme activity cannot be tested by CMCNa assay alone, we tested CenA and Cex migrated solution using FPA (Filterd paper assay), incubated at 50℃, pH=7. Nevertheless, the results was unsatisfying due to FPA assay is not a sensitive measure method and secretory enzyme activity was relatively low. Besides, time was limited for us for we constructed positive clone just before deadline, we failed to obtain cex secretive enzyme activity.
Some suggestions: cex gene is abundant of C and G, it's hard to be amplified by PCR without smear or unspecific band, it would be better if you synthesis the gene directly in your targeted plasmid.
References
[1]Duedu KO, French CE. Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass. Enzyme Microb Technol. 2016 Nov;93-94:113-121. doi: 10.1016/j.enzmictec.2016.08.005. Epub 2016 Aug 8. PubMed PMID: 27702471.
[2]Su L, Chen S, Yi L, Woodard RW, Chen J, Wu J. Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3). Microb Cell Fact. 2012 Jan 12;11:8. doi: 10.1186/1475-2859-11-8. PubMed PMID: 22239833; PubMed Central PMCID: PMC3286373.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 596
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 54
Illegal XhoI site found at 63 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 229
Illegal NgoMIV site found at 602
Illegal NgoMIV site found at 1104 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 649
Illegal SapI.rc site found at 732
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