Intermediate

Part:BBa_K299812

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

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

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. 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.





Function of inv-llo gene on the platform of cyanobacteria

Author: Chiu Hsun-I from NYMU.Taipei, 2012

 Introduction
References revealed when engineered with invasin(inv) from Yersinia pestis and listeriolysin O(llo) from Listeria monocytogenes[1], new S. elongatus was able to invade into cultured mammalian cells and was capable of symbiosis with eukaryotic cells. The symbiosis possibility of induced pluripotent stem cells from mice and J774 mouse macrophage cell line were evaluated.</p>

Method and materials Plasmid preparation

Invasin(inv) from Yersinia pestis and listeriolysin O(llo) from Listeria monocytogenes were provided by Pamela A. Silver, Harvard Medical school[1].

Symbiosis experiment

Three types of mammalian cells were evaluated—J774 mouse macrophage cell line and mouse induced pluripotent stem cells in embryonic stem(ES) cells stage and embryoid body(EB) stage.Synechococcus elongatus PCC 7942 was the first cyanobacterial strain which could be transformed by exogenously added DNA.Engineered cyanobacteria which were transformed inv-llo plasmid cocultured with cells under Leibovitz's L-15 medium without antibiotics. After 24 hours, supernatants were discarded and cells were washed with PBS two times and the medium was replaced by L-15 containing antibiotics. Cells were fixed by 4% paraformaldehyde and were observed by fluorescent microscope or confocal laser microscope after 24 hours. Detailed methods were described on NYMU.Taipei wiki.

 Discussion and Results
Under the fluorescence microscope 530-550 nm excitation filter, red pots were S. elongates PCC7942. Under the transmitted light, round and transparent one was cell.

Figure 1 revealed wildtype and inv-llo transformed type both entered into J774 macrophage cell line by phagocytosis and the help of inv-llo.

J774 invllo.jpg inv-llo strain, merged image of fluorescence and bright field<p>

J774 wt.jpg wildtype, merged image of fluorescence and bright field


Figure 2 showed the coculture results of iPS cells at ES stage. Results showed that inv-llo transformation would increase the recruitment of cyanobacteria on induced pluripotent stem cells instead of the entry.

IPS invllo.jpg inv-llo strain, merged image of fluorescence and bright field.

IPS wt.jpg wildtype, merged image of fluorescence and bright field.


On figure 3, there were similar and relative results of the iPS cells at EB stage to iPS cells at ES stage. The symbiosis of stem cells would be more difficult but the recruitment provided a potential for invading mammalian cells.

EB invllo.jpg inv-llo strain, merged image of fluorescence and bright field


 Conclusion
The symbiosis possibility of induced pluripotent stem cells from mice and J774 mouse macrophage cell line were evaluated. Results showed that invasin and listeriolysin O transformation would raise the entry speed and escape rate in J774 mouse macrophage cell line, and would increase the recruitment of cyanobacteria on induced pluripotent stem cells.


 References

[1]Towards a Synthetic Chloroplast, Christina M. Agapakis,1 Henrike Niederholtmeyer,1,¤ Ramil R. Noche,1 Tami D. Lieberman,1 Sean G. Megason,1 Jeffrey C. Way,2 and Pamela A. Silver1,2,PLoS One. 2011; 6(4): e18877.



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
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
n/aInvasin + Listeriolysin