Team:Paris/Transduction overview

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<span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Transduction_overview#top | Receiving the message]] > [[Team:Paris/Transduction_overview#bottom | Membrane fusion]]
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== Overview ==
 
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'''Aim:'''
 
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- to enable gene transcription after fusionning OMVs with the outer membrane of the receiving bacterium.  
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==Membrane fusion: Main==
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We also hope that we could achieve this aim without sacrifying important proprieties of our message : specific, repeatable , multidirectional.
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It seems that we have two possible ways : the ABC transporters or the two component systems .
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ABC transporters and two component systems are natural transport system (export or import) of information, nutriments or toxines.
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<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Transduction_overview#bottom"> Main </a>|
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Transduction_overview_fusion#bottom"> Fusion</a>|
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Transduction_overview_strategy#bottom"> Our strategy</a>|
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Transduction_overview_construction#bottom"> Construction</a>
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There is another possibility but the mecanism is mostly unknown : the DNA-containing OMVs which could be a useful mean of information transport.
 
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This part of the project was focus on a precise point
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*[[Team:Paris/Transduction_overview_fusion#bottom |The fusion between the OMVs and the targeted bacteria.]]
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----
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We have planned to explore three different method :
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'''ABC transporter'''
 
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----
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'''''With the Jun/Fos dimere:'''''
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Jun and Fos are able to form an heterodimer which has a high stability and Jun can dimerize with another Jun (thanks to their leucine zipper motif).
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After mutations into the leucine zipper motif of Jun (that allow the Jun/Fos dimerization but avoid the Jun/Jun homodimer formation), we wanted to fuse it to AIDA (an ABC transporter) to send them to the extern membrane of bacteria.
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'''''With G3P :'''''
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The viral protein known as G3P is naturally exposed at the surface of the filamentous bacteriophage which enable it to get in the bacteria. The M13 phage has a high affinity for E.coli, and if we could place its G3p on the surface of the vesicles it could activate the fusion with the Outer membrane of the targeted bacteria.
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<u>Overview:</u>
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In order to target the G3P at the surface of of the vesicles, we fuse it to the OmpA- Linker protein (created by the Warsaw team)
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The ABC transporter is a major class of cellular translocation machinery  encoded in the largest set of paralogous genes.
 
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The ABC (ATP binding cassette) transporter is one of the active transport systems of the cell,
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'''''With the SNARE system:'''''
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which is widespread in archaea, eubacteria, and eukaryotes. It is also known as the
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Intracellular membrane fusion in eukaryotes requires SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins that form complexes bridging the two membranes. To allow this fusion, the perfect conformation of all protein that composed the SNARE complex is an obligation. Untill now, no one cloned this complex into bacteria. We thought that it will be a very "dangerous way" to go for our project, so we decided to focus our effort on the Jun/Fos strategy and the G3P one.
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periplasmic binding protein-dependent transport system in Gram-negative bacteria and the binding-  
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lipoprotein-dependent transport system in Gram positive bacteria. The transporter shows a common
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global organization with three types of molecular components. Typically, it consists of two integral membrane proteins (permeases)
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each having six transmembrane segments, two peripheral membrane proteins that bind and hydrolyze ATP,
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and a periplasmic (or lipoprotein) substrate-binding protein. The ATP-binding protein component is the most
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conserved, the membrane protein component is somewhat less conserved, and the substrate-binding protein
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component is most divergent in terms of the sequence similarity. The ABC transporters form the largest group of paralogous genes in bacterial
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and archaeal genomes , and the genes for the three components frequently form an operon.  
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the following scheme shows a typical ABC mechanism :
 
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<u>uses:</u>
 
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transport the protein directly into the cytolpasm to activate the transcription.
 
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<u>advantages/drawbacks:</u>
 
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advantages:
 
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the protein of interest is directly translocated in the cytoplasm and if it is a transcription factor it could activated immediatly the response.
 
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drawbacks:
 
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It is a nutriment uptake system, so basically only small molecules are able to pass throught the membranes and it is a very specific.
 
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<u>the trick :</u>
 
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Few ABC transporter such as FecABCD (iron transporter) are able to induce à response regardless of the tranlocation, due to the activity of FecA, some mutant can also have a constitutive expression of FecABCD . We could use FecA- mutant  receiver
 
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and FecA+ mutant donor to transfert working FecA to the receiver. In this case the receiver will express the FecABCD operon, and so we could place under the control of the Fec ABCD promoter the gene sequence encoding for the response.
 
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'''problems :''' the message is unidirectionnal and unrepeatable. It would just be a proof of principle that a vesicle-mediated controlled communication is possible.
 
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'''Two-component system'''
 
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<u>overview:</u>
 
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The TCS can be cosidered as a widely spread class of biosensor knowing that adaptive signal transduction within
 
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microbial cells involving a multi-faceted regulated phosphotransfer mechanism that comprises structural rearrangements
 
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of sensor histidine kinases upon ligand-binding and phosphorylation-induced conformational changes in response
 
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regulators of versatile two-component systems (TCS), arisen early in bacterial evolution.
 
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In most eubacteria, two-component proteins typically constitute *1% of encoded peptides. In
 
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pathogenic bacteria they control the expression of important pathogenetic factors, in addition to regulating basic
 
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housekeeping functions. The widespread distribution of two-component signal transduction systems in Bacteria and
 
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Achaea reflect their biological value as major sensing and response elements to a wide range of environmental insults
 
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that are tuned to respond from within milliseconds to hours . Although TCSs are probably the most
 
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efficient means of adaptation to conventional stressful stimuli encountered by bacteria during their lifespan, the
 
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plasticity of some of these sophisticated systems may contribute to strain-specific cellular processes and to the
 
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acquisition of distinct features and phenotypes, particularly in pathogens .
 
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To put the structure in a nutshell :
 
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A typical TCS consists of a transmembrane dimeric sensor histidine kinase (HK) and a cytoplasmic cognate response regulator (RR).
 
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In gram negative bacteria there is often a Periplasmic Binding Protein which optimize the dectection of the molecule localized in the periplasm
 
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by a high affinity for the HK after binding the specific molecule.
 
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the following scheme shows a typical ABC mechanism :
 
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<u>uses:</u>
 
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By transferring a packet of molecule synthesize by the donnor but not present in the medium,
 
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the arrival could activate the transcription of gene of interest.
 
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<u>advantages/drawbacks:</u>
 
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advantages:
 
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This mecanism is dedicated to the transription of gene under a specific promoter, the difficulty is to find an easily exportable and detectable signal.
 
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drawbacks:
 
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The biosensor are generaly sensible to high diffusible molecule for which the vesicles transport is useless.
 
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<u>the trick:</u>
 
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We could with the help of Alfonso Jaramillo design a synthetic PBP which could detect the substrate we choosed and activate a specific HK.
 
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The previous work (Valencia 2006) design a vanilin-sensitive PBP and a network for a graduated response whereas we just need a proteic sensitive PBP and a binary type of response.
 
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strain and part needed:
 
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strain : E.coli MRi7 (unable to translocate ribose from periplasm to cytoplasme)
 
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part :
 
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==référence :==
 
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*1995- Cosima Harle & Volkmar Braun - Signal transfer through three compartments transcription initiation of the Escherichia coiferric  citrate transport system from the cell surface
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*2000 - Stock & Goudreau – Two-component signal  transduction
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*2001 - Mishima & Murata - Super-channel in bacteria  function and structure of the macromolecule import system mediated by a pit-dependent ABC transporter
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*2009 - Akyriakidis & Tiligada – signal transduction TCS  the AtoSC paradigm
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*2009 - Tomii & Kanehisa – comparative analysis of ABC transporter
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Latest revision as of 15:06, 21 October 2009

iGEM > Paris > Receiving the message > Membrane fusion



Membrane fusion: Main


This part of the project was focus on a precise point

We have planned to explore three different method :


With the Jun/Fos dimere: Jun and Fos are able to form an heterodimer which has a high stability and Jun can dimerize with another Jun (thanks to their leucine zipper motif). After mutations into the leucine zipper motif of Jun (that allow the Jun/Fos dimerization but avoid the Jun/Jun homodimer formation), we wanted to fuse it to AIDA (an ABC transporter) to send them to the extern membrane of bacteria.


With G3P : The viral protein known as G3P is naturally exposed at the surface of the filamentous bacteriophage which enable it to get in the bacteria. The M13 phage has a high affinity for E.coli, and if we could place its G3p on the surface of the vesicles it could activate the fusion with the Outer membrane of the targeted bacteria.

In order to target the G3P at the surface of of the vesicles, we fuse it to the OmpA- Linker protein (created by the Warsaw team)


With the SNARE system: Intracellular membrane fusion in eukaryotes requires SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins that form complexes bridging the two membranes. To allow this fusion, the perfect conformation of all protein that composed the SNARE complex is an obligation. Untill now, no one cloned this complex into bacteria. We thought that it will be a very "dangerous way" to go for our project, so we decided to focus our effort on the Jun/Fos strategy and the G3P one.


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