Difference between revisions of "Part:BBa K3661007"

(→‎Characterization)
(→‎Characterization)
 
Line 15: Line 15:
 
[[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]]
  
−
[[File:T--CPU_CHINA--K3661007_p2.png|600px|thumb|center|'''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.]]
+
[[File:T--CPU_CHINA--K3661007_p2.png|600px|thumb|center|'''Figure 2.''' Result of activity assay using human flag ELISA kit.]]
  
−
[[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>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.</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>
+
 
 +
[[File:T--CPU_CHINA--K3661007_p3.png|600px|thumb|center|'''Figure 3.'''Comparison of Corrected A<sub>450</sub> of Sample Groups.]]
 +
 
 +
<p>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.</p>
  
 
<p>JM79 was added from 5.5h. The group that added JM79 showed decreased growth relative to the negative control (added the supernatant of uninduced <i>E.coli</i> ).</p>
 
<p>JM79 was added from 5.5h. The group that added JM79 showed decreased growth relative to the negative control (added the supernatant of uninduced <i>E.coli</i> ).</p>

Latest revision as of 19:31, 27 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.

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]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