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Team:Berkeley/Project/Construction
From 2012.igem.org
We chose to use Golden Gate Assembly because it allowed us to either create single-pot libraries or the interchange of combinatorially expand the number of multi-gene devices with smaller constituent parts. Golden Gate Assembly is powered by Type IIs restriction enzymes, which have many key features that should be mentioned.
- They cut distal to their recognition sites. Because of this, we have full control over the resulting 4bp overhangs, giving us a theoretical choice between 4^4, or 256 different overhangs. In practice, we reduced this set, eliminating cases where at 2/4 bases matched and palindromes, to minimize chances of mis-annealing.
- They have directionality. Because the cutting site is not the recognition site (#1), the cutting can either happen to 5' or 3' to the recognition site. This can be modulated by reversing the sequence
An example type IIs restriction enzyme, BsmBI.
In classic Golden Gate Assembly, these type IIs enzymes together with T4 DNA Ligase to glue together these overhangs. Starting with either a 1) PCR product or a 2) plasmid , the assembly can be done in two ways:
A) Sequentially with an interstitial gel purification step, or
B) In a single reaction that oscillates between digestions and ligations to move towards a correct product. Because type IIs endonucleases cut distal to their recognition site, once they form their .
In our MiCode Assembly (MCA), we utilized two type IIs enzymes, BsaI and BsmBI. By alternating the usage of these two enzymes, we can build up our combinatorial set of unique devices in every round of cloning. In this section, we will show how we designed MiCode Assembly interchangeability and iterative set expansion that allow MiCodes
The basic, fundamental unit of our GGA is the part plasmid, shown below. This is synonymous with the part plasmids kept in the registry, but instead of being flanked with EcoRI, XbaI, SpeI, and PstI, it is flanked by an outer BsmBI and an inner BsmBI.
