Team:MIT mammalian Standard

From 2010.igem.org

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<ul>
<ul>
                         <li><a href="http://2010.igem.org/Team:MIT_toggle">Overview</a></li>
                         <li><a href="http://2010.igem.org/Team:MIT_toggle">Overview</a></li>
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                        <li><a href="http://2010.igem.org/Team:MIT_tmodel">Modelling</a></li>
<li><a href="http://2010.igem.org/Team:MIT_tconst">Toggle Construction</a></li>
<li><a href="http://2010.igem.org/Team:MIT_tconst">Toggle Construction</a></li>
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<li><a href="#">Characterization</a></li>
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<li><a href="http://2010.igem.org/Team:MIT_composite">Characterization</a></li>
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</dd>
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</ul>
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<dl id="specialnav">
<dt><b>Mammalian</b></dt>
<dt><b>Mammalian</b></dt>
<dd>
<dd>
<ul>
<ul>
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                         <li><a href="#">Overview</a></li>
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                         <li><a href="http://2010.igem.org/Team:MIT_mammalian">Overview</a></li>
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<li><a href="#">Standard and Design</a></li>
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<li><a href="http://2010.igem.org/Team:MIT_mammalian_Standard">New Mammalian Standard </a></li>
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<li><a href="#">Experiments</a></li>
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                        <li><a href="http://2010.igem.org/Team:MIT_mammalian_Circuit">Circuit Design</a></li>
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<li><a href="http://2010.igem.org/Team:MIT_mammalian_Mechanosensation"> Mechanosensation</a></li>
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<li><a href="http://2010.igem.org/Team:MIT_mammalian_Bone"> Bone Formation</a></li>
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<li><a href="http://2010.igem.org/Team:MIT_mammalian_Switch"> Synthetic Switch</a></li>
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<div id="unique" style="padding:0px; font-size: 14px; border: 1px solid black; margin:0px; background-color:transparent;">
<div id="unique" style="padding:0px; font-size: 14px; border: 1px solid black; margin:0px; background-color:transparent;">
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<table width=650px style="background-color: white; margin-top:5px; padding: 10px;"><tr><td><div class="bodybaby">Motivation </div></td>
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<table width=650px style="background-color: white; margin-top:5px; padding: 10px;"><tr><td><div class="bodybaby">New Mammalian Assembly Standard </div></td>
<tr><td>
<tr><td>
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Mammalian promoters are difficult to biobrick. They're several kB in length, much longer then their prokaryotic counterparts. This means they're likely to contain most restriction sites used in biobrick cloning. It's difficult to avoid this by changing the promoter sequence; single base pair mutations often alter or abolish the desired function of the promoter. To get around this issue, we've created a new standardization for cloning in mammalian cells, based on the Invitrogen Gateway (c) cloning system.  
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Mammalian promoters are difficult to biobrick. They're several kB in length, much longer then their prokaryotic counterparts. This means they're likely to contain most restriction sites used in biobrick cloning. It's difficult to avoid this by changing the promoter sequence; single base pair mutations often alter or abolish the desired function of the promoter. To get around this issue, we've created a new standardization for cloning in mammalian cells, based on the Invitrogen Gateway (c) cloning system. <A HREF="http://2010.igem.org/wiki/images/3/3e/Mammoblock_RFC_Draft.pdf">Read our full 'MammoBlock' standardization proposal</A>
<br><br>
<br><br>
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Gateway (c) is a fast and reliable way to create expression vectors for mammalian cells. It fulfills the same function as restriction cloning in the Biobrick standardization - it allows us to combine vectors with different 'parts' to create a whole 'circuit'. But the actual mechanism is vastly different from restriction cloning. Gateway uses recombination enzymes to combine multiple vectors, a one-step process that avoids the laborious digestion and ligation steps involved in restriction cloning.
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<table><tr><td>
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<br> <br>
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<a href="http://2010.igem.org/wiki/images/e/ed/Gateway_image.tiff" class="thickbox" title="Multi-site LR Gateway Reaction"><img src="http://2010.igem.org/wiki/images/e/ed/Gateway_image.tiff" width=300px></a></td><td>
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<b>Cloning Process</b>
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Gateway (c) is a fast and reliable way to create expression vectors for mammalian cells. It fulfills the same function as restriction cloning in the Biobrick standardization - it allows us to combine vectors with different 'parts' to create a whole 'circuit'. But the actual mechanism is vastly different from restriction cloning. Gateway uses recombination enzymes to combine multiple vectors, a one-step process that avoids the laborious digestion and ligation steps involved in restriction cloning.</td></tr></table>
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<br>
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We start out by cloning all the genes and promoters needed into pENTR vectors; the pENTR vectors contain restriction and recombination sites, so either cloning method can be used to insert the target DNA into the vectors. The next step is to combine pENTR vectors containing the relevant gene and promoter with a pDEST vector containing a lentiviral origin of replication. Gateway cloning allows us to avoid laborious digestion and ligation steps in favor a faster, more efficient method. To obtain the expression vector, we combine all three plasmids in a recombination reaction; the step takes 12-16 hours total and yields remarkably reliable products.
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<b>Cloning Process Overview</b>
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<br>
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<b>Gateway Recombination Mechanism</b>
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We start out by cloning all the genes and promoters needed into pENTR vectors; the pENTR vectors contain restriction and recombination sites, so either cloning method can be used to insert the target DNA into the vectors. The next step is to combine pENTR vectors containing the relevant gene and promoter with a pDEST vector containing a lentiviral origin of replication. Gateway cloning allows us to avoid laborious digestion and ligation steps in favor of a faster, more efficient method. To obtain the expression vector, we combine all three plasmids in a recombination reaction; the step takes 12-16 hours total and yields remarkably reliable products.
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Two recombination reactions form the basis for Gateway cloning. The enzymes involved function in the lysogenic cycle of the temperate lambda bacteriophage. During the lysogenic cycle, lambda bacteriophage integrates its genome into the host E. coli genome. This allows the bacteriophage to lie dormant for several generations, until the lytic cycle is again activated. The enzymes that control this integration are integrase (Int), excisionase (Xis) and integration host factor (IHF). Integrase can catalyze either excision or integration of the genome; IHF binds Int and catalyzes the binding affinity for the recombination sites by manipulating the structure of the DNA.
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<br><br>
<br><br>
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The recombination sites involved are termed attB, attP, attL and attR; during integration, Int catalyzes the recombination of attB and attP sites to form an attL and attR sites flanking the integrated genome. The opposite reaction takes place for excision; the Int catalyzes the recombination of the attL and attR sites, yielding a liberated phage genome with an attP site, and the original host genome with an attB site (see below). The reaction equilibrium normally favors the attB and attP recombination. The excisionase Xis catalyzes excision by binding to the attR site and shifting the equilibrium to favor the reverse reaction.
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<b>Our Contribution</b>
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<br>
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We are in the process of adding two new backbones to the registry; pENTRL4R1 is a backbone for promoter parts (see map below). The location of the attL4 and attR1 recombination sites place the promoter in front of the gene of interest during Gateway recombination. This construct contains a TRE-inducible promoter between the recombination sites.
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<br>
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<center>
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<a href="http://2010.igem.org/wiki/images/0/0b/L4_TRE_R1_image.jpg" class="thickbox" title="MammoBlock Promoter Backbone"><img src="http://2010.igem.org/wiki/images/0/0b/L4_TRE_R1_image.jpg" width=400px></a></center>
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<br>
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The second backbone designed to support gene 'parts'. It contains attL1 and attL2 recombination sites flanking the gene of interest.  
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<br>
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<center>
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<a href="http://2010.igem.org/wiki/images/3/33/L1_EGFP_L2_image.jpg" class="thickbox" title="MammoBlock Gene Backbone"><img src="http://2010.igem.org/wiki/images/3/33/L1_EGFP_L2_image.jpg" width=400px></a></center>
</td>
</td>

Latest revision as of 01:07, 28 October 2010

Mammalian
New Mammalian Assembly Standard
Mammalian promoters are difficult to biobrick. They're several kB in length, much longer then their prokaryotic counterparts. This means they're likely to contain most restriction sites used in biobrick cloning. It's difficult to avoid this by changing the promoter sequence; single base pair mutations often alter or abolish the desired function of the promoter. To get around this issue, we've created a new standardization for cloning in mammalian cells, based on the Invitrogen Gateway (c) cloning system. Read our full 'MammoBlock' standardization proposal

Gateway (c) is a fast and reliable way to create expression vectors for mammalian cells. It fulfills the same function as restriction cloning in the Biobrick standardization - it allows us to combine vectors with different 'parts' to create a whole 'circuit'. But the actual mechanism is vastly different from restriction cloning. Gateway uses recombination enzymes to combine multiple vectors, a one-step process that avoids the laborious digestion and ligation steps involved in restriction cloning.

Cloning Process Overview
We start out by cloning all the genes and promoters needed into pENTR vectors; the pENTR vectors contain restriction and recombination sites, so either cloning method can be used to insert the target DNA into the vectors. The next step is to combine pENTR vectors containing the relevant gene and promoter with a pDEST vector containing a lentiviral origin of replication. Gateway cloning allows us to avoid laborious digestion and ligation steps in favor of a faster, more efficient method. To obtain the expression vector, we combine all three plasmids in a recombination reaction; the step takes 12-16 hours total and yields remarkably reliable products.

Our Contribution
We are in the process of adding two new backbones to the registry; pENTRL4R1 is a backbone for promoter parts (see map below). The location of the attL4 and attR1 recombination sites place the promoter in front of the gene of interest during Gateway recombination. This construct contains a TRE-inducible promoter between the recombination sites.

The second backbone designed to support gene 'parts'. It contains attL1 and attL2 recombination sites flanking the gene of interest.