Part:BBa_K1897013

Listeriolysin O

Safety Flag

The iGEM Safety and Security Committee has placed a Red Flag on this part. This part presents safety risks beyond what is normal for the Registry. Researchers who plan to acquire and use this part should take special care to ensure they use it safely and responsibly. Contact safety [AT] igem [DOT] org with any questions.

Reason: Listeriolysin and Invasin parts

If you are an iGEM team, you must submit a Check-In before acquiring and using this part! See the 2021 Safety Page for more information.

BBa_K1897013

This part was made up of promoter pLuxR BBa_R0062, ribosome binding site (RBS) BBa_B0030, Listeriolysin O (LLO) coding sequence BBa_K1897012 and double terminator BBa_B0015. It is derived from Listeria monocytogenes and is a pore-forming toxin that allows L. monocytogenes to escape from the endocytic vesicle into the cytoplasm (Vadia et al., 2011).

Usage and Biology

LLO is essential for the pathogenesis of L. monocytogenes and is part of the family of the cholesterol-dependent cytolysis (CDCs). They have a size range of 50 to 70 kDa, and bind to a host receptor on the host membrane. Upon binding, they diffuse laterally to form a ring-shaped oligomeric prepre complex then then inserts a large β-barrel pore across the membrane in a cholesterol-dependent fashion (Vadia et al., 2011). Formation of the pore allows LLO to escape from the phagosomal compartment that it is in when it first enters a cell.

LLO also has a acidic pH optimum (around pH 5 to 5.5), which may thus explain why it is able to escape the phagosomal compartment that it is trapped in when it first enters the cell, but requires additional internalisation proteins for it to first enter a non-phagocytic cell (Kayal and Charbit, 2006; Vadia et al., 2011).

This part can be used in conjunction with BBa_K1897011 to allow for entry into a mammalian cell and allow a bacterium to escape into the cytosol of a mammalian cell.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1482
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 90
  
• 1000
  COMPATIBLE WITH RFC[1000]

Construction and verification of composite LLO

LLO was sent for synthesis, and the transformed Escherichia coli colonies were miniprepped and RE-digested with EcoRI and PstI to identify those with the synthesized plasmid (Figure 1). The expected band size was around 1895 bp, which is likely to be the bands that are boxed up in Figure 1.

Figure 1: Gel electrophoresis photo for the screening of positive colonies with the composite LLO plasmid. A restriction enzyme (RE) digestion was performed using EcoRI and PstI on the individual plasmids. The expected band size for LLO was around 1895 bp and it can be seen in the black box. Each lane in the box is a duplicate digest reaction.