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
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 CenA solution.
The CMCNa assays were incubated at 37°C and pH7. Based on literature, In a microtube 18μL of 1% CMC-Na was added, followed by 2μL crude CenA and the mix was incubated for 1 hour. To stop and reveal the reaction, 30 μL of DNS were added, after 1 hour of 99°C incubation, absorbance at 540 nm was determined. For negative control, we performed contrast reactions at the same time, but it was reacted at the temperature below 4°C so that the enzyme activity was inhibited.
Figure 2. supernatant enzyme activity of cenA-hlyA/hlyBhlyD/pIN2
The supernatant was concentrated by 20 times when performing CMCNa assay, but the enzyme activity had been adjusted to standard unit
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 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