Part:BBa_K1897013
Listeriolysin O
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1482
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 90
- 1000COMPATIBLE 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 ___ and ___ to identify those with the synthesized plasmid (Figure 1).
INSERT FIGURE 1 (LLO RE digest screening)
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