Part:BBa_K1054002

bacteriophage PBSX holin

Holin is an essential protein for host lysis by bacteriophage. This part is cloned from Bacillus subtilis subsp. subtilis str. 168. Its activity can by blocked by another protein called lrgA (BBa_K1054024) and lrgB (BBa_K1054003). We can use this feature to prevent horizontal gene transfer of certain plasmid.

Growth curve of BL21 E.coli cells induced by different concentrations of IPTG

Contribution: iGEM22_WHU-China

PBSX holin is a small membrane protein in double-stranded DNA phages for efficient host lysis. Previous research has found that Holins are the gatekeepers of the lysis process, possessing an intriguing ability to be triggered, at a precise time point, to form large holes in the cytoplasmic membrane of phage-infected bacteria. ([1] Mart Krupovič, Dennis H. Bamford. 2008)

Figure. 1 The ‘death raft’ model for holin lesion formation. (Mart Krupovič, Dennis H. Bamford. 2008)

In this research, a ‘death raft’ model is used to describe holin lesion formation. The process includes accumulation and oligomerization of holins in the CM, formation of the ‘death raft’, the opening of an aqueous channel and formation of the holin lesion. (Figure. 1)

After understanding the functional mechanism of holin, we predicted holin’s structure by PolyPhobius prediction. (https://phobius.sbc.su.se/poly.html) It shows that PBSX holin has two transmembrane regions. (Figure. 2)

Figure. 2 The prediction of the transmembrane domain of PBSX holin.

Inspired by the properties of holin mentioned above, we think holin is an effective and secure choice for our kill switch, for it will cause the cleavage of the engineered bacteria only, releasing no toxin in the gut or environment.

We induced the expression of PBSX holin in E. coli at the log phase and measured OD600. The result shows the ability of holin to inhibit the growth of bacteria to some extent. (Figure. 2) Reference:

Figure. 3: Induction of the expression of PBSX holin in E. coli. A.Induction on a culture plate. B. Change in turbidity.

Reference:
[1] Mart Krupovicˇ and Dennis H. Bamford. Holin of bacteriophage lambda: structural insights into a membrane lesion. Molecular Microbiology(2008)69(4), 781–783.

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]