Part:BBa_K2609017

T4 endolysin under a constitutive promoter

This generator expresses the T4 endolysin gene (a phage lysozyme) under BBa_J23106, a medium strength constitutive promoter. This part was used to screen for recombined phages after Lambda Red recombination.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 311
  Illegal AgeI site found at 381
  
• 1000
  COMPATIBLE WITH RFC[1000]

Biology

The T4 Bacteriophage uses a holin-endolysin system for host lysis. Endolysin, which has no secretory signal sequence, accumulates in a fully folded and active state in the cytosol. Holin, a late stage phage protein, triggers the degradation of the cell membrane allowing Endolysin to reach its target, the cell wall. In the phage genome, Endolysin is encoded by the e-gene and e^- mutants have been shown to lack any noticeable lytic activity. Expression of holin without a corresponding endolysin results in complete irreversible cessation of host metabolism without any structural damage to the outer membrane (thus preventing lysis).

Usage

We used this part for screening of recombined phages lacking the e (endolysin) gene after Lambda Red recombination. The T4 phage strains without the e-gene that codes for Endolysin were modified using homologous recombination to add mcp-1 attached to signal peptide under the t gene(codes for holin).The recomninant T4 phages give very small or no plaques on performing a plaque assay on the wild strains of E. coli as opposed to the wild type T4 phages. However on replica plating on the E.coli cells having the part transformed into them will give plaques with Wild type as well as the recombinant T4 phages. Thus we were able to select for the recombined phages.^[1][2]

Characterization

Expression and characterization

The part was transformed into E.coli BL21(DE3). We ran an SDS-PAGE of the cell lysate of the transformed clones against the protein ladder.The protein has a size~18.7 kDa. However due to the presence of E.coli proteins of similar size, there is no significant difference seen in the two lanes^[3]. We characterized the protein by checking for its lytic activity as mentioned below.

Characterisation of lystic activity of Endolysin

The lytic activity of endolysin was characterised by measuring the decrease in the optical density of B.cereus cell suspension after addition of endolysin^[4][5]. Exponentially growing Bacillus cereus cells were washed twice and resuspended in 0.2mM Tris-HCl(pH-8) to adjust to OD_600nm= 0.6-0.8. Then Endolysin protein(100 µL) was added to 900µL of cell suspension. The following were used as controls:
1. 900µL cell suspension with 100µL of resuspension buffer.
2. 900µL cell suspension with 100µL of Lysis Buffer used in protein extraction.
3. 900µL cell suspension wiht 100µL of Proteins from Wild Type E.coli(BL21(DE3))
The OD_600nm at 37^o was measured using a plate reader at regular intervals of time and the lytic activity was noted from the Change in OD v/s time curve as in Figure 2.

Addition of Endolysin has a much stronger lytic activity compared to the buffer alone. The higher activity of endolysin than the proteins extracted from the wild type led us to the conclusion that the lytic activity is conferred due to the Endolysin plasmid inserted.

References

[1]Eric S. Miller, Elizabeth Kutter,et al."Bacteriophage T4 Genome". Microbiol. Mol. Biol. Rev. Mar 2003, 67 (1) 86-156; DOI: 10.1128/MMBR.67.1.86-156.2003<br [2]Wang, I N et al. “Holins: the protein clocks of bacteriophage infections.” Annual review of microbiology 54 (2000): 799-825. [3]Lim, Jeong-A YangᆞSoon-Ryun et al. “Exogenous lytic activity of SPN9CC endolysin against gram-negative bacteria.” Journal of microbiology and biotechnology 24 6 (2014): 803-11.
[4]Jaeeun Park, Jiae Yun, et al. "Characterization of an endolysin, LysBPS13, from a Bacillus cereus bacteriophage", FEMS Microbiology Letters, Volume 332, Issue 1, 1 July 2012, Pages 76–83, https://doi.org/10.1111/j.1574-6968.2012.02578.x
[5]Lim, Jeong-A, et al. "Characterization of Endolysin From a Salmonella Typhimurium-infecting Bacteriophage Spn1s." Research in microbiology 163.3 (2012): 233-241. doi: 10.1016/j.resmic.2012.01.002