Composite

Part:BBa_K3661005

Designed by: Weixuan Lu   Group: iGEM20_CPU_CHINA   (2020-10-23)
Revision as of 19:31, 27 October 2020 by Hongfa (Talk | contribs) (→‎Characterization)

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This composite part is composed of secretion peptide YebF, bacteriocin Hiracin JM79 and fusion tag FLAG. Peptide YebF is added to achieve the secretion of bacteriocin Hiracin JM79 into intestinal environment or culture supernatant. YebF secretion system is widely used in E.coli. [1] Hiracin JM79 has been tested and reported to have activity against enterococcus faecalis.[2] A major advantage of the Hiracin JM79 as an antimicrobial agent is its specificity compared to many traditional antibiotics.

Usage

In 2020 CPU_CHINA project, YebF, Hiracin JM79 and FLAG were co-expressed to form the fused protein, the attachment of YebF peptide make it possible for JM79 to be secreted out of the chasis and achieve certain concentration in intestinal environment, thus be able to kill Entrococcus faecalis properly. By reducing the quantity of E.faecalis, the balance of intestinal flora can be restored thus alleviate the inflammation induced by enterococcus faecalis.

Biology

YebF secretion system is widely used in escherichia coli. Bacteriocins linked to the carboxyl end of YebF are efficiently secreted. Hiracin JM79 (HirJM79), a Sec-dependent bacteriocin produced by Enterococcus hirae DCH5, was proved to have activity against enterococcus faecalis with certain specificity.

Characterization

We constructed a secretory expression vector for YebF-Hiracin JM79-flag, using escherichia coli as chasis. YebF-Hiracin JM79-flag was detected using an ELISA kit.

Figure 1.Standard curve of flag protein.
Figure 2.Result of activity assay using human flag ELISA kit.

CFUs were inoculated to LB medium followed by overnight incubation at 37℃. After induction of IPTG, the culture was incubated at 16℃ for 17h. Cells and medium were collected and ready for assay.

Figure 3.Comparison of Corrected A450 of Sample Groups.

It showed that the Hiracin JM79 expression of the positive group induced by IPTG was significantly higher than that of the uninduced negative group. The sample group was significantly different compared to the negative control group.*P < 0.05, ** P < 0.01, *** P < 0.001, and ****P < 0.0001 by t test.

JM79 was added from 5.5h. The group that added JM79 showed decreased growth relative to the negative control (added the supernatant of uninduced E.coli ).

We tested the antibacterial effect of hiracin JM79. As shown in Figure 4, the quantity of Enterococcus faecalis in the experimental group induced by 5h was significantly lower than that of the negative control group, which proved that our system can successfully secrete The bacteriocin also achieves the bactericidal effect on Enterococcus faecalis.

Figure 4.The killing assay of E.faecalis by JM79.

Reference

[1]Zhang, G., Brokx, S., & Weiner, J. H. (2006). Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in Escherichia coli. Nature biotechnology, 24(1), 100–104. https://doi.org/10.1038/nbt1174

[2]Sánchez, J., Diep, D. B., Herranz, C., Nes, I. F., Cintas, L. M., & Hernández, P. E. (2007). Amino acid and nucleotide sequence, adjacent genes, and heterologous expression of hiracin JM79, a sec-dependent bacteriocin produced by Enterococcus hirae DCH5, isolated from Mallard ducks (Anas platyrhynchos). FEMS microbiology letters, 270(2), 227–236. https://doi.org/10.1111/j.1574-6968.2007.00673.x

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