GoldenGate reactions are entirely based off of their part definitions, which are 4 base pair overhangs which are exposed when cut with a Type IIS restriction enzyme, particularly BtgZI, BsaI, BsmBI, or BbsI. To ensure an efficient and precise reaction, most scientists recommend that all of the definitions should not have 2 or more base pairs in the same position to minimize non-specific ligation.
However, having set definitions is also a disadvantage when assembling parts in different orders. Unlike biobricks, which can be assembled modularly in any order, GoldenGate parts are usually stuck in the definitions the scientist cloned for them. BtgZI shuffle is a solution to this problem, allowing more modular assembly of multi part biosynthesis systems.
In BtgZI shuffle, definitions are swapped. As seen in Fig.1, the blue definitions are swapped for the red ones. The blue ones are still inside the sequence, but Type IIS restriction enzymes no longer act on them, so they are considered simply ‘scar sequences’. The new part definitions, in red, can be acted upon by Type IIS enzymes, and therefore are the active overhang sequences. This allows for additional GoldenGate reactions.
Since this is essentially a simple GoldenGate reaction, it is recommended to use current high-efficiency protocols.
Alternatively, a single step 5 minute reaction would also be possible (9)(10)(11). Simply place DNA in mastermix, wait 5 minutes, then transform into E coli using the plate n’ go procedure.
However, some scientists complain (12) that GoldenGate assembly using this method would take far too long. The solution is taking advantage of T5 exonuclease to chew back linear DNA ends after the original reaction. After this, purify the reaction and directly use in a PCR. After the PCR, cut with BsaI-HF and DpnI and gel purify. Place directly into a GoldenBraid reaction and transform.
Keoni Gandall, 3:31 18.2.15
References:
A. Ravikumar 12
