Difference between revisions of "Part:BBa K299812"

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	<b>Bacteria transformed with this part are invasive microorganisms.</b> 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.</p>		<b>Bacteria transformed with this part are invasive microorganisms.</b> 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.</p>
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	<p><h3>Function of inv-llo gene on the platform of cyanobacteria </h3>		<p><h3>Function of inv-llo gene on the platform of cyanobacteria </h3>

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Revision as of 03:58, 26 September 2012

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.

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 (both provided by Pamela A. Silver, Harvard Medical school[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.

 Method and materials

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.Cells were seeded on the 24-well plate. After 80% full in each well, PCC7942(wt/inv-llo transform) suspensions in PBS were set to the same OD and 50 µl of this suspension were added per 1 ml of Leibovitz's L-15 medium without phenol red, without antibiotics and with 10% fetal bovine serum (Invitrogen) per well of 24-well tissue culture dishes containing the mammalian cells. After 24-hour cells and PCC7942 coculturing under 30°C atmospheric CO2 level, supernatants were discarded and cells were washed with PBS two times and the medium was replaced by L-15 containing antibiotics, gentamycin 100ug/ml(Invitrogen). After 24 hours, cells were fixed by 4% paraformaldehyde and were observed by fluorescent microscope or confocal laser microscope.

[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