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Team:MIT mammalian Standard
From 2010.igem.org
iGEM 2010
Motivation |
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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. 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 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 promoters 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 |
