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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.
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. Gateway Recombination Mechanism 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. 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. |
