Difference between revisions of "Part:BBa K3034004"

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<partinfo>BBa_K3034004 short</partinfo>
 
<partinfo>BBa_K3034004 short</partinfo>
  
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This lysin is formed by fusion of lysep3 and D8,which lyses bacteria from inside and outside the cell.The bactericidal spectrum of lysep3-D8 is broad, as it can lyse both gram-negative bacteria and gram-positive bacteria(14 E. coli strains, 3 P. aeruginosa strains, 1 Acinetobacter baumannii strain,and 1 Streptococcus strain).
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This lysin is formed by fusion of lysep3 and D8,which lyses bacteria from inside and outside the cell.The bactericidal spectrum of lysep3-D8 is broad, as it can lyse both gram-negative bacteria and gram-positive bacteria(14 <i>E. coli</i> strains, 3 <i>P. aeruginosa</i> strains, 1 <i>Acinetobacter baumannii</i> strain,and 1 <i>Streptococcus</i> strain) [1].
  
 
===Usage and Biology===
 
===Usage and Biology===
  
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To achieve lytic function, we usually express Lysep3-D8 protein. Combination of the inducible promoter with this part allows expression under specific conditions to inhibit the growth of ''E. coli''. We can also introduce it into ''E. coli'' BL21 (DE3) strain expression and use it as an "antibiotics" when a sufficient amount of protein is obtained.
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To achieve lytic function, we usually express Lysep3-D8 protein. Combination of the inducible promoter with this part allows expression under specific conditions to inhibit the growth of ''E. coli'' BL21(DE3).  
  
 
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===Characterization===
 
===Characterization===
  
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In order to verify the lytic effect of Lysep3-D8 protein, we cloned the corresponding coding gene into the vector induced by IPTG, and then induced the expression of this gene by adding IPTG with a final concentration of 0.5mM to obtain the intracellular lysis effect of Lysep3-D8 against ''E.coli'' DH5α(culture temperature 37 ̊C). At the same time, in order to verify the extracellular cleavage of ''E.coli'' BL21(DE3), we transformed the plasmid into ''E.coli'' BL21(DE3) and induced expression at 16 ̊C (Final concentration of IPTG was 0.5mM) for 16 hours to obtain a sufficient amount of protein. After that, we added the protein to E. coli DH5α cultured to logarithmic growth phase to verify the effect of extracellular lysis.
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In order to verify the lytic effect of Lysep3-D8 protein, we cloned the corresponding coding gene into the vector induced by IPTG, and then induced the expression of this gene by adding IPTG with a final concentration of 0.5mM to obtain the intracellular lysis effect of Lysep3-D8 against ''E.coli'' BL21(DE3)(culture temperature 37 ̊C).  
  
 
===Experiment Results===
 
===Experiment Results===
  
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We showed the growth of ''E. coli'' by measuring the growth curve of the Abs600 value of the bacterial liquid every hour. The results are as follows:
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We showed the growth of ''E. coli'' BL21(DE3) by measuring the growth curve of the OD600 value of the bacterial liquid every hour. The results are as follows:
  
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[[File:T--UESTC-China--safety1.png|700px|thumb|center|'''Fig.1''' Characterization of intracellular cleavage effect of Lysep3-D8 protein(37˚C). The experimental data comes from two sets of biological replicates, repeated three times in each group.]]
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[[File:T--UESTC-China--safety1.png|700px|thumb|center|'''Fig. 1.''' Characterization of intracellular cleavage effect of Lysep3-D8 protein(37˚C) in ''E.coli'' BL21(DE3). The experimental data comes from two sets of biological replicates, repeated three times in each group.]]
  
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Figure 1 shows that when the expression of Lysep3-D8 was induced by the addition of IPTG at a final concentration of 0.5 mM, the difference in Abs600 between the experimental group (+IPTG) and the control group (-IPTG) was larger and larger. That is, the Lysep3-D8 protein inhibited the growth of Escherichia coli (BL21) (inhibition rate was about 30.7%).
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Our experiments results(Fig. 1) showed that when the expression of Lysep3-D8 was induced by the addition of IPTG at a final concentration of 0.5 mM, the difference in OD600 between the experimental group (IPTG+) and the control group (IPTG-) was obvious. That is, the Lysep3-D8 protein inhibited the growth of ''E.coli'' (BL21) (inhibition rate was about 30.7%).
  
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===Reference===
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[1] Shuang Wang, Jingmin Gu, Meng Lv, et al. (2017). The antibacterial activity of E. coli bacteriophage lysin lysep3 is enhanced by fusing the Bacillus amyloliquefaciens bacteriophage endolysin binding domain D8 to the C-terminal region. <p style="display: inline;font-style: italic"> Journal of Microbiology,</p> 55:403<br>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 01:33, 22 October 2019


The fusion protein is an enhanced lysin-> lysep3-D8

This lysin is formed by fusion of lysep3 and D8,which lyses bacteria from inside and outside the cell.The bactericidal spectrum of lysep3-D8 is broad, as it can lyse both gram-negative bacteria and gram-positive bacteria(14 E. coli strains, 3 P. aeruginosa strains, 1 Acinetobacter baumannii strain,and 1 Streptococcus strain) [1].

Usage and Biology

To achieve lytic function, we usually express Lysep3-D8 protein. Combination of the inducible promoter with this part allows expression under specific conditions to inhibit the growth of E. coli BL21(DE3).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 79
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

In order to verify the lytic effect of Lysep3-D8 protein, we cloned the corresponding coding gene into the vector induced by IPTG, and then induced the expression of this gene by adding IPTG with a final concentration of 0.5mM to obtain the intracellular lysis effect of Lysep3-D8 against E.coli BL21(DE3)(culture temperature 37 ̊C).

Experiment Results

We showed the growth of E. coli BL21(DE3) by measuring the growth curve of the OD600 value of the bacterial liquid every hour. The results are as follows:

Fig. 1. Characterization of intracellular cleavage effect of Lysep3-D8 protein(37˚C) in E.coli BL21(DE3). The experimental data comes from two sets of biological replicates, repeated three times in each group.

Our experiments results(Fig. 1) showed that when the expression of Lysep3-D8 was induced by the addition of IPTG at a final concentration of 0.5 mM, the difference in OD600 between the experimental group (IPTG+) and the control group (IPTG-) was obvious. That is, the Lysep3-D8 protein inhibited the growth of E.coli (BL21) (inhibition rate was about 30.7%).

Reference

[1] Shuang Wang, Jingmin Gu, Meng Lv, et al. (2017). The antibacterial activity of E. coli bacteriophage lysin lysep3 is enhanced by fusing the Bacillus amyloliquefaciens bacteriophage endolysin binding domain D8 to the C-terminal region.

Journal of Microbiology,

55:403