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Materials and Methods
From 2008.igem.org
Materials and Methods
1. Cloning
Constructs were cloned by the AarI method. This a multi-part/combinatorial cloning method that is particularly well suited to shuffling protein domains with various promoters and terminators. The key to this approach is the Type II-S restriction enzyme, AarI, a rare (7-cutter) that cuts 4bp offset from its binding site. Thus, AarI can generate four base overhangs of any sequence.
Since the user can specify the overhangs, this method can be used to "stitch-together" fragments without a scar, which is sometimes necessary to preserve protein function. More importantly, these overhangs can be non-palindromic, which solves the biggest problem faced when trying to do multipart ligations using standard restriction enzymes, illustrated here:
This allows high efficiency ligations using up to 4 parts (vector plus 3 inserts). While parts can be made with any 4 base overhang (end), we chose a standard set, termed A, B, C, and D.
These ends yield 3 possible parts: AB, BC and CD. For two part ligations, we use AB and BD parts. Parts are typically generated by PCR from a genomic DNA or plasmid template, then TOPO cloned and sequenced:
Digestion yields parts with standard, non-palindromic, overhangs.
AarI Shuttle Vector To facilitate exchange of parts between AarI users and the biobricks community, we are offering a Shuttle Vector. This vector accepts AarI parts in the AB or BD format (as were used in our 2008 project). These parts can then be cut out of the vector, with in-frame biobrick ends.
