Team:MIT mammalian Standard

<li><a href="#">Characterization</a></li>

                         <li><a href="#">Overview</a></li>

<li><a href="#">Standard and Design</a></li>

<li><a href="#">Experiments</a></li>

<table width=650px style="background-color: white; margin-top:5px; padding: 10px;"><tr><td><div class="bodybaby">Motivation </div></td>

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.  

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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<b>Cloning Process</b>

 

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.

 

 

 

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.