Composite

Part:BBa_K299814

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

invasin listeriolysin GFP

Invasin (INV) plays a key role in the initiation of Yersinia enterocolytica and Yersinia pseudotuberculosis infection. Through interaction with a beta1-integrin receptor present on the surface of eucaryotic cell membranes it triggers a signal-transduction pathway leading to internalisation of the whole bacterium in the endocytosis-dependent manner. The strong affinity of INV to it’s receptor results in highly selective binding to the target molecule. Mammalian cells depleted of beta1-integrin cannot be infected.

Listeriolysin O (LLO) 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

Green Fluorescent Protein is a noninvasive fluorescent marker for gene expression, protein localisation and intracellulat protein targeting. Originally from Aequorea victoria.

Authors:

Cloned by Joanna Leszczyńska.
Created with the use of parts BBa K299810 and BBa K299811 cloned by Marta Błaszkiewicz.

Work supervised by Michał Lower at all levels.

Construct design.

The part consists of B0032 RBS ligated to inv gene from Yersinia pestis (horizontal gene transfer form Yersinia pseudotuberculosis). Following elements are B0032 RBS ligated to 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. GFP as marker. Double terminator (B0010+B0012) guarantees the stability of polycistronic RNA as a product of transcription. 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.

Bacteria transformed with this part are invasive microorganisms. 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 3936
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 770
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1387
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 4819


[edit]
Categories
Parameters
n/ainvasin listeriolysin GFP