Team:MIT mammalian Standard

From 2010.igem.org

(Difference between revisions)
Line 60: Line 60:
<tr><td>
<tr><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.  <A HREF="http://2010.igem.org/wiki/images/3/3e/Mammoblock_RFC_Draft.pdf">Read our full 'MammoBlock' standardization proposa</A>  
+
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>
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.
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.

Revision as of 02:49, 26 October 2010

Motivation
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

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.