Team:Paris/Production overview Construction

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{{Template:Paris2009_menu2}}
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==Overview==
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* [[Team:Paris/Production_overview#Overview | Introduction]]
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==Construction==
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* [[Team:Paris/Production_overview_Tol/Pal#Overview |A. Tol/pal ]]
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* [[Team:Paris/Production_overview_strategy#Overview |B. Our strategy]]
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* [[Team:Paris/Production_overview_Construction#Overview |C. Construction]]
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Production_overview#bottom"> Main </a>|
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<a class="menu_sub" href="https://2009.igem.org/Team:Paris/Production_overview#Vesicle_production_system_:_Tol.2FPal"> Tol/Pal</a>|
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Production_overview#Vesicle_production_system_:_Our_strategy"> Our strategy</a>|
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<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Production_overview_Construction#bottom"> Construction</a>
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===Creation of OMVs===
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==C. Construction==
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To create vesicles, we started with a first simple construction only containing the nonfunctional TolR II biobrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_K257005 Bba K257005] fused with OmpA signal [http://partsregistry.org/wiki/index.php?title=Part:BBa_K257006 Bba_K257006] (which allows it to migrate in the periplasm) placed donwstream  a ptet promoter [https://2009.igem.org/Team:Paris/constructions#tolr (Freezer)]. In the presence of arabinose, the TetR repressor is expressed from the pLac promoter and inhibits the expression of tolRII (Figure a). The addition of tetracycline or of one of its analog, aTc, in the medium relieves the repression thus allowing the production of vesicles (Figure b).
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===C.1. Creation of OMVs===
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To create vesicles, we started with a first simple construction only containing the nonfunctional TolR II biobrick placed donwstream a ptet promoter<b class="correction"> [liens vers la construction - Nom/Freezer] </b>. In the presence of arabinose, the TetR repressor is expressed from the pBad promoter and inhibits the expression of tolRII (Figure a). The addition of tetracycline or of one of its analog, aTc, in the medium relieves the repression thus allowing the production of vesicles (Figure b).
 
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<b class="correction"> images à refaire hein mon cricri...</b>
 
<center><b><big>Production of vesicles OFF</big></b></center>
<center><b><big>Production of vesicles OFF</big></b></center>
[[Image:Production ON.jpg|750px|center]]
[[Image:Production ON.jpg|750px|center]]
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For further precisions on the biobricks used as well as the plasmid backbones, please visit our [https://2009.igem.org/Team:Paris/Production_design parts design page ].
For further precisions on the biobricks used as well as the plasmid backbones, please visit our [https://2009.igem.org/Team:Paris/Production_design parts design page ].
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===C.2. The delay===
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<span/ id="1">
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===The delay===
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Our aim is to send vesicles containg proteins (the "message" in the bubble) inside the medium and we want to wait for the proteins to be present inside the periplasm or on the outer membrane before  creaing vesicles.
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To avoid a premature production of vesicles, we have introduced a delay system inside the plasmids<b class="correction">[Liens - Nom/freezer] </b>. This kind of device was studied by Hooshangi & Weiss <b class="correction">[Références] </b>using a transcriptional cascade made of two repressor namely TetR and LacI :
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Our aim is to send vesicles containing proteins (the "message" in the bubble) into the medium and.
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To avoid a premature production of "empty" vesicles, we want to wait for the proteins to be present inside the periplasm or in the outer membrane before  creaing vesicles. We have introduced a delay system inside the plasmids [https://2009.igem.org/Team:Paris/constructions#ptet (Freezer)]. This kind of device was studied by Hooshangi & Weiss<sup>[[https://2009.igem.org/Team:Paris/Production_overview_Construction#References 1]]</sup> where they studied a transcriptional cascade made of two repressor: TetR and LacI.
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Nevertheless, as the pLacI promoter is leaking <b class="correction">mettre une ref</b>, we tried to exchange the order of the two repressors to avoid a low but constant expression of TolR. When there is arabinose in the medium, the production of LacI represses the Plac promoter and prevents the creation of TetR ; this way, the Ptet promoter is no longer repressed and GFP is expressed in the medium.
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Nevertheless, as in this construction, the pLacI promoter is leaking, we decided to exchange the order of the two repressors to avoid a low but constant expression of TolR. When there is arabinose in the medium, the production of LacI represses the Plac promoter and prevents the creation of TetR; this way, the Ptet promoter is no longer repressed and GFP is expressed in the medium.
With this design, even if there is some leaky expression of the genes downstream the pLac promoter (when it is repressed), it leads to the creation of TetR and a stronger repression of the pTet promoter.
With this design, even if there is some leaky expression of the genes downstream the pLac promoter (when it is repressed), it leads to the creation of TetR and a stronger repression of the pTet promoter.
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We also decided to place the pTet+TolRII device upstream the pLac+TetR cassette <b class="correction">[Liens - Nom/freezer] </b>; as a consequence, even if our first  terminator is not a 100% efficient, the RNA-polymerase will transcribe the downstream part made of the TetR repressor. This repressor created by accident will then repress the expression of TolRII.
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We also decided to place the pTet+TolRII device upstream the pLac+TetR cassette [https://2009.igem.org/Team:Paris/constructions#tolr2 (Freezer)]; as a consequence, even if our first  terminator is not a 100% efficient, the RNA-polymerase will transcribe the downstream part made of the TetR repressor. This repressor created by accident will then repress the expression of TolRII.
If we had switched these two parts, a bad efficiency of the terminator would have led to the expression of more TolRII which could be dangerous for the cell.
If we had switched these two parts, a bad efficiency of the terminator would have led to the expression of more TolRII which could be dangerous for the cell.
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These considerations led us to construct this system :
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These considerations led us to construct this final system :
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[[Image:Global Off.jpg|800px|center]]
[[Image:Global Off.jpg|800px|center]]
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<center>When there is no more arabinose in the medium, TetR is expressed and the pTet promoter repressed, stoping the vesicules production.</center>
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<center>When there is no more arabinose in the medium, TetR is expressed and the pTet promoter repressed, stopping vesicles production.</center>
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*To read more on terminators, you can also have a look at the registry: [http://partsregistry.org/Help:Terminators Terminators.]''
*To read more on terminators, you can also have a look at the registry: [http://partsregistry.org/Help:Terminators Terminators.]''
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===C.3. Caracterisation of the delay===
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===Caracterisation of the delay===
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===C.4. The LVA tag===
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===The LVA tag===
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<span /id="2">
The LacI protein is supposed to be quite a stable protein in the cells ; as a way to reduce its half life and thus to decrease the strenght of the plac repression (see modelling part for analytic interpretation), we decided to use a LVA tag on the LacI protein.
The LacI protein is supposed to be quite a stable protein in the cells ; as a way to reduce its half life and thus to decrease the strenght of the plac repression (see modelling part for analytic interpretation), we decided to use a LVA tag on the LacI protein.
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The LVA tag is a short peptide sequence added to the C-terminal end of a nucleotide sequence ; in the periplasm, degradation of  AANDENYALAA-tagged proteins is known to be the result of the Tsp protese whereas in the cytoplasm, it is due to a Tsp homolog. Mutations in the last three residues of the AANDENYALAA tail alter the protein stability.  
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The LVA tag is a short peptide sequence added to the C-terminal end of a nucleotide sequence ; in the periplasm, degradation of  AANDENYALAA-tagged proteins is known to be the result of the Tsp protese whereas in the cytoplasm, it is due to a Tsp homolog. Mutations in the last three residues of the AANDENYALAA tail alter the protein stability<sup>[[https://2009.igem.org/Team:Paris/Production_overview_Construction#References 2]]</sup>.  
These different mutations were studied by Andersen & Molin on the Gfp protein both in E.Coli and P.Putida. In both species, protein including a LVA mutation were the most rapidly degraded. These mutations were also used by Ellowitz and Leibler in the repressilator device in order to obtain an efficient oscillator.
These different mutations were studied by Andersen & Molin on the Gfp protein both in E.Coli and P.Putida. In both species, protein including a LVA mutation were the most rapidly degraded. These mutations were also used by Ellowitz and Leibler in the repressilator device in order to obtain an efficient oscillator.
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==References==
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New Stable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria
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<ol class="references">
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<li> [[Team:Paris/Production_overview_Construction#1 | ^]]Ultrasensitivity and noise propagation in a synthetic transcriptional cascade S.Hooshangi & R.Weiss
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2005  - [http://www.ncbi.nlm.nih.gov/pubmed/15738412 15738412]</li>
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</div>
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<li> [[Team:Paris/Production_overview_Construction#2 | ^]]New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria Jens Bo Andersen & Søren Molin 1998  - [http://www.ncbi.nlm.nih.gov:80/pmc/articles/PMC106306/ 106306]</li>
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====References====
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[x] <b class="correction">Folaf 'aire la référence/indexation </b>
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Latest revision as of 19:36, 21 October 2009

iGEM > Paris > Vesicle production system > Constructions


Contents

Construction

Creation of OMVs

To create vesicles, we started with a first simple construction only containing the nonfunctional TolR II biobrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_K257005 Bba K257005] fused with OmpA signal [http://partsregistry.org/wiki/index.php?title=Part:BBa_K257006 Bba_K257006] (which allows it to migrate in the periplasm) placed donwstream a ptet promoter (Freezer). In the presence of arabinose, the TetR repressor is expressed from the pLac promoter and inhibits the expression of tolRII (Figure a). The addition of tetracycline or of one of its analog, aTc, in the medium relieves the repression thus allowing the production of vesicles (Figure b).


Production of vesicles OFF
Production ON.jpg


Production of vesicles ON
Production OFF.jpg



For further precisions on the biobricks used as well as the plasmid backbones, please visit our parts design page .

The delay

Our aim is to send vesicles containing proteins (the "message" in the bubble) into the medium and. To avoid a premature production of "empty" vesicles, we want to wait for the proteins to be present inside the periplasm or in the outer membrane before creaing vesicles. We have introduced a delay system inside the plasmids (Freezer). This kind of device was studied by Hooshangi & Weiss[1] where they studied a transcriptional cascade made of two repressor: TetR and LacI.


Initial Transcriptional Cascade
Figure a


Nevertheless, as in this construction, the pLacI promoter is leaking, we decided to exchange the order of the two repressors to avoid a low but constant expression of TolR. When there is arabinose in the medium, the production of LacI represses the Plac promoter and prevents the creation of TetR; this way, the Ptet promoter is no longer repressed and GFP is expressed in the medium.

With this design, even if there is some leaky expression of the genes downstream the pLac promoter (when it is repressed), it leads to the creation of TetR and a stronger repression of the pTet promoter.


We also decided to place the pTet+TolRII device upstream the pLac+TetR cassette (Freezer); as a consequence, even if our first terminator is not a 100% efficient, the RNA-polymerase will transcribe the downstream part made of the TetR repressor. This repressor created by accident will then repress the expression of TolRII.

If we had switched these two parts, a bad efficiency of the terminator would have led to the expression of more TolRII which could be dangerous for the cell.


These considerations led us to construct this final system :


Final Construction ON
Global On.jpg
When arabinose is placed in the medium, the pBad promoter is ON and allows the creation of LacI ; LacI represses the pLac promoter and the TetR protein is no longer expressed. The pTet promoter is derepressed and TolRII triggers the production of vesicules.



Final Construction OFF
Global Off.jpg
When there is no more arabinose in the medium, TetR is expressed and the pTet promoter repressed, stopping vesicles production.



Notes :

  • To get more information on the terminators we use, you can see the description in the registry for:
    • [http://partsregistry.org/Part:BBa_B0014 BBa_B0014]
    • [http://partsregistry.org/Part:BBa_B0015 BBa_B0015.]
  • To read more on terminators, you can also have a look at the registry: [http://partsregistry.org/Help:Terminators Terminators.]

Caracterisation of the delay

We have started the construction of a system which would allow us to characterize the delay created by our device. In this construction, instead of expressing the proteins we want to send inside the vesicules and the TolRII protein, we express the [http://partsregistry.org/Part:BBa_E0030 YFP] and the RFP respectively. Using fluorescent microscopy, we can try to measure this delay.


As shown on the figure below, once there is arabinose in the medium, the pBad promoter is ON allowing the creation of LacI ; LacI represses the pLac promoter and the TetR protein is no longer expressed. The pTet promoter is ON and RFP is created :



Delay Test ON
Figure a



On the other hand, when there is glucose in the medium (and no Arabinose), LacI is no longer produced and the TetR protein is created : there is no more RFP synthetized :



Delay Test OFF
Figure a


The LVA tag

The LacI protein is supposed to be quite a stable protein in the cells ; as a way to reduce its half life and thus to decrease the strenght of the plac repression (see modelling part for analytic interpretation), we decided to use a LVA tag on the LacI protein.

The LVA tag is a short peptide sequence added to the C-terminal end of a nucleotide sequence ; in the periplasm, degradation of AANDENYALAA-tagged proteins is known to be the result of the Tsp protese whereas in the cytoplasm, it is due to a Tsp homolog. Mutations in the last three residues of the AANDENYALAA tail alter the protein stability[2].

These different mutations were studied by Andersen & Molin on the Gfp protein both in E.Coli and P.Putida. In both species, protein including a LVA mutation were the most rapidly degraded. These mutations were also used by Ellowitz and Leibler in the repressilator device in order to obtain an efficient oscillator.

References

  1. ^Ultrasensitivity and noise propagation in a synthetic transcriptional cascade S.Hooshangi & R.Weiss 2005 - [http://www.ncbi.nlm.nih.gov/pubmed/15738412 15738412]
  2. ^New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria Jens Bo Andersen & Søren Molin 1998 - [http://www.ncbi.nlm.nih.gov:80/pmc/articles/PMC106306/ 106306]

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