Difference between revisions of "Part:BBa K299813"

Revision as of 22:03, 26 October 2010

This part is licensed under
Creative BioCommons

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 listeriolysin 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.

model LLO
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 AraC promoter and B0032 RBS ligated to inv gene from Yersinia pestis (horizontal gene transfer form Yersinia pseudotuberculosis). Following elements are B0032 RBS ligated to synthetic listeriolysin gene originally from Listeria monocytogenes. GFP as marker. Double terminator (B0010+B0012) guarantees the stability of polycistronic RNA as a product of transcription. The construct is a full Invasiveness Operon. 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.

Performance
Microscopic observation of HeLa cells infected with BactoDHL confirmed the functionality of the system.

FOTO inv+lis_259
HeLa cells incubated with E.coli strain harbouring Invasiveness Operon. Thanks to FRET between Hirsch dye and GFP infected cells can be seen as a mildly-glowing areas containing bright spots of GFP inside. For more photos visit Gallery of Microphotographs.

Flow Cytometry made it possible to express in numbers what could be seen under the microscope. For each sample during preperatics lysozyme was used to prevent the adhesion of bacteria to the surface of HeLa, but 100% efficiency cannot be expected form this procedure (see HeLa Cells incubated with GFP-producing but non-invasive Top10) The results of control experiments are as follows:.............

</p>

Sequence and Features