Part:BBa_K2609017

T4 endolysin under a constitutive promoter

This generator expresses the T4 endolysin protein (a phage lysozyme) under BBa_J23106, a medium strength constitutive promoter. Endolysin acts against the peptidoglycan layer and is minimally toxic to bacterial cells when produced internally because of the presence of an internal lipid membrane. An exposure of the peptidoglycan layer to endolysin however results in its disruption. Gram negative bacteria are resistant to lysis induced by external endolysin because of the presence of an outer membrane.

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 and irreversible cessation of host metabolism without any structural damage to the outer membrane.^[1]

Usage

The 2018 IISc-Bangalore iGEM team used this part for screening of recombined phages lacking the e (endolysin) gene after Lambda Red recombination. The e-gene in the phage genome was replaced with a non-cognate protein and this part was used to screen for recombinants .The recombinant T4 phages give very small or no plaques on performing a plaque assay on the wild strains of E. coli (because of improper lysis) as opposed to the wild type T4 phages. However, replica plating of the plaques on E.coli cells having this part transformed into them allows easy identification by checking the plaques that have increased in size from one plate to another. The presence of recombinants can then be verified by a plaque PCR with primers for the non-cognate sequence.

Characterization

Expression and characterization

The part was transformed into E.coli BL21(DE3) and an SDS-PAGE of the cell lysate was performed.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^[2]. 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^[3][4]. Exponentially growing Bacillus cereus cells were washed twice and resuspended in 0.2mM Tris-HCl(pH-8) to adjust the 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 Abs_600nm at 37^oC was measured using a plate reader at regular intervals of time and the lytic activity was quantified from the Change in %Abs v/s time curve as in Figure 2.

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
[2]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.
[3]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
[4]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