Intermediate

Part:BBa_K299811

Designed by: Michał Lower   Group: iGEM10_Warsaw   (2010-10-13)


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Safety Flag

The iGEM Safety 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 2017 Safety Page for more information.


This part is licensed under
Creative BioCommons

B0032 Listeriolysin


Listeriolysin O is a member of a widespread cholesterol-dependent pore-forming cytolysins family (CDCs). It's natural role in Listeria monocytogenes is to provide endosomal escape. The first step of the process involves binding of monomeric listeriolysin molecules to lipid bilayer containing cholesterol. The binding induces conformational change that subsequently leads to the formation of a prepores' oligomeric structures (consisting of 33-50 monomers) converting into large (maximum 350A-diameter) pores. This severely disturbs the stability of endosomal membrane and causes it’s rupture.

LLO is a phagosome-specyfic lysin. The acidic pH is necessary for it’s full hemolytic activity. Neutral pH of cytosol causes premature unfolding of TMH domains responsible for aqueous pore formation. This mechanism prevents Listeria spp from killing the host cell and losing the intracellular environment. In case of any tranformed strain it guarantees the lowest possible level of cytotoxicity, incomparable to this involved with the use of any other protein from CDCs family.

Left: Structural model of the LLO monomer from: P. Schnupf , D.A. Portnoy Listeriolysin O: a phagosome-specific lysin, Microbes and infection (2007) 1176-1187

Authors:

Cloned by Marta Błaszkiewicz under supervision of Michał Lower.

Construct design.

The part consists of B0032 RBS followed by synthetic gene encoding listeriolysin, codon usage optimized for E. coli. Aminoacid sequence identical with mature form of LLO from Listeria monocytogenes. Original signal sequence (secretion signal) is omitted since it does not work in E. coli. The construct is a fragment of the Invasiveness Operon (BBa_K299813 and BBa_K299815) To find out more about it's background and design click here.

Safety.

All safety precautions must be taken when manipulating with transformed strain. It involves obligatory use of laboratory gloves. Work under laminar is strongly advised. All waste should be autoclaved to avoid accidental gene transfer to other bacteria and potential rise of pathogenic organism.






Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1399
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
n/aB0032 Listeriolysin