Difference between revisions of "Part:BBa K3661007"
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===Characterization===
 
===Characterization===
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<p>Plasmid (YebF-Hiracin JM79-flag) was transfected into <i>E.coli BL21(DE3</i>). Hiracin JM79 was secreted from the engineered bacteria through YebF secretion system. In order to determine whether Hiracin JM79 was successfully expressed in the cellular environment , we performed Elisa of flag protein. Then we calculated the amount of Hiracin JM79 based on the expression of flag protein.</p>
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<p>Plasmid (YebF-Hiracin JM79-flag) was transfected into <i>E.coli BL21(DE3</i>). Hiracin JM79 was secreted from the engineered bacteria through YebF secretion system. In order to determine whether Hiracin JM79 was successfully expressed in the cellular environment , we performed ELISA of flag protein. Then we calculated the amount of Hiracin JM79 based on the expression of flag protein.</p>
  
 
[[File:T--CPU_CHINA--K3661007_p1.png|600px|thumb|center|'''Figure 1.'''Standard curve of flag protein]]
 
[[File:T--CPU_CHINA--K3661007_p1.png|600px|thumb|center|'''Figure 1.'''Standard curve of flag protein]]
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[[File:T--CPU_CHINA--K3661007_p3.png|600px|thumb|center|'''Figure 3.'''Comparison of Corrected A<sub>450</sub> 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]]
 
[[File:T--CPU_CHINA--K3661007_p3.png|600px|thumb|center|'''Figure 3.'''Comparison of Corrected A<sub>450</sub> 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]]
 
<p>The sample group was significantly different compared to the negative control group.*P &lt; 0.05, ** P &lt; 0.01, *** P &lt; 0.001, and ****P &lt; 0.0001 by t test.</p>
 
<p>The sample group was significantly different compared to the negative control group.*P &lt; 0.05, ** P &lt; 0.01, *** P &lt; 0.001, and ****P &lt; 0.0001 by t test.</p>
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===Reference===
 
===Reference===
 
<p>[1]Sánchez, J., Diep, D. B., Herranz, C., Nes, I. F., Cintas, L. M., &amp;  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). <em>FEMS microbiology letters</em>, <em>270</em>(2), 227–236. https://doi.org/10.1111/j.1574-6968.2007.00673.x</p>
 
<p>[1]Sánchez, J., Diep, D. B., Herranz, C., Nes, I. F., Cintas, L. M., &amp;  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). <em>FEMS microbiology letters</em>, <em>270</em>(2), 227–236. https://doi.org/10.1111/j.1574-6968.2007.00673.x</p>

Revision as of 12:56, 26 October 2020

Hiracin JM79 have been tested and reported to have activity against enterococcus faecalis.[1] A major benefit of the Hiracin JM79 as an antimicrobial agent is their specificity compared to many traditional antibiotics.


Usage

In 2020 CPU_CHINA project, Hiracin JM79 were used to kill entrococcus faecalis. Hiracin JM79 can be secreted from engineered bacteria to increase the extracellular concentration. Bacteriocins thus improve ALD in patients by killing enterococcus faecalis in the cellular environment.


Biology

Bacteriocins are proteinaceous or peptidic toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain(s). Hiracin JM79 (HirJM79), a Sec-dependent bacteriocin produced by enterococcus hirae DCH5, was proved to have activity against enterococcus faecalis.


Characterization

Plasmid (YebF-Hiracin JM79-flag) was transfected into E.coli BL21(DE3). Hiracin JM79 was secreted from the engineered bacteria through YebF secretion system. In order to determine whether Hiracin JM79 was successfully expressed in the cellular environment , we performed ELISA of flag protein. Then we calculated the amount of Hiracin JM79 based on the expression of flag protein.

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.

Reference

[1]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