Device

Part:BBa_K299813

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

AraC regulated cell invasion device


Bacteria transormed with this part are able to enter the cytosol of 48,82% of exposed mammalian cells.


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 invasin 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 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 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 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


Right: 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 are as follows:





Left: HeLa Cells incubated with non-invasive, non-transformed Top10 E. coli – no fluorescence observed;
Right: HeLa Cells incubated with GFP-producing but non-invasive Top10. Hardly noticeable fluorescence observed.

Right: HeLa Cells incubated with AraC+INV+LLO+GFP-transformed E. coli Top10.


Two different “populations” of GFP can be observed, depending on two varying fluorescence levels. These represent the amounts of protein (indicated by marker M2 on histograms) still entrapped in endosome and (indicated by marker M3 on histograms) distributed to cytoplasm. This interpretation is based on the results of many previous studies indicating that the GFP is able to act as a pH sensor.

The existence of relationship between pH and levels of GFP fluorescence is a commonly known fact. The Microfluidic system was used to investigate this relationship on the FACS machine used for Invasiveness Operon testing.



The data analysis leads to a simple conclusion: The less acidic (closer to neutral) the pH, the stronger the GFP fluorescence. This strongly supports our theory: in the lyzosome (pH~5) weaker fluorescence of GFP is observed than in the cytosol (pH~7).


HeLa cells incubated with non-transformed Top10, GFP-producing Top10 and AraC Invasiveness Operon-transformed E. coli Top 10 compared to J23102 Invasiveness Operon-harbouring Top10 performance: percentage of GFP delivered to different compartments.


Please note the high levels of invaded cells (86,74% and 87,04% for J23102 and pAraC constructs, accordingly). In reference to control experiments (Top10), it has been proven that results up to 10% can be explained by spontaneous uptake. The result for HeLa incubated with GFP-producing, noninvasive Top 10 is explained by both spontaneous uptake and minor adhesion not eliminated by lysozyme.


Efficiency of GFP delivery to mammalian cells cytoplasm


Bacteria transormed with this part are able to enter the cytosol of 48,82% of exposed mammalian cells.

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 4093
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 927
    Illegal BamHI site found at 103
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1544
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 4976


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Categories
Parameters
n/aAraC regulated cell invasion device