Part:BBa_K3034004

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).

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. 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.

Experiments

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.

Results

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:

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.

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%).

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]