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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 not palindromic, and thus have directionality. Because the recognition site is non-palindromic, the cutting can either happen to 5' or 3' to the recognition site. This can be modulated by reversing the sequence.
- They can cut themselves out. Depending on the direction of cutting, the site can be cut out of the desired fragment so that it does not undergo digestion after it has been ligated.
- Some are compatible with DNA ligase buffer.
An example type IIs restriction enzyme, BsmBI. In this diagram, a GACC overhang is created.
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.
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 a connection with the correct neighbor, the product is fixed.
In our MiCode Assembly (MCA), we utilized two type IIs enzymes, BsaI and BsmBI. By alternating the usage of these two enzymes in each round of assembly and reintroducing the sites when necessary, we can combine parts . (In this section, we will show how we designed MCA to emphasize interchangeability that allows for iterative set expansion which enables us to efficiently build 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 outer BsaI sites. These BsaI sites create unique overhangs when digested, which anneal with other complementary overhangs in a single-pot reaction. Conveniently, BsaI and BsmBI are compatible with T4 DNA Ligase, allowing for the two to be combined in a single pot reaction that cycles between optimal temperatures for digestion (37˚C) and ligation (16˚C). Because correct products are fixed, the system converges towards our final product. We use plasmids because they allow us to easily amplify and sequence the DNA.
Each part is flanked by unique overhangs that dictate its potential neighbors, its position in the cassette, and the type of part it is (detailed below).
A part plasmid coding for PAmCherry, our photoactivatible red fluorescent protein.
Several part plasmids can be joined via their overhangs to produce a cassette. There are several unique positions within a cassette:
#1: 5' region.
#2: Promoter.
#3: Protein, which can be split into 3a/3b for protein fusions. For example, we linked the protein that localized to actin, nucleus, and vacuolar membrane (part 3a) to a fluorescent protein (part 3b).
#4: Terminator.
#5: 3' region.
#6: Markers and origins of replication. Can be split into 6a/6b to include both E. coli and yeast markers.
Keep in mind that for each position in the cassette, there are multiple parts with compatible overhangs (each position has multiplicity), which allows us to easily interchange parts from our library of parts we and the Dueber Lab have made. In this cassette assembly step, all the components of this cassette are physically linked together. The physical linkage retains the data about this unit of the MiCode in such a way that we can utilize it downstream. This allows us to treat the entire cassette as a single unit and enables us to build larger constructs using the cassettes with the confidence that the genotype remains stable.
A cassette coding for nucleus-localized PAmCherry with ConS and Con2 connector regions.
In the next round of assembly, several cassettes are combined together to form multigene cassettes. Because the multigene cassettes build off of the cassettes, they retain their information. At each position between connector regions, there are multiple cassettes we can substitute in to create our desired MiCode. This modularity enables us to build a large combinatorial set of MiCodes with only a few cassettes.
A multigene cassette coding linking various MiCode components to a leucine zipper. This half codes for the bait zipper.
MiCode components linked to a prey zipper.
Up until this point, we have been building one construct per reaction because we wanted to know precisely what we were building. After we build a library of bait and a separate library of prey zippers by hand, we can combine the two together in a large single-pot reaction. Because we designed the bait half-code to only be compatible with the prey half-code, we can ensure. Additionally, because all components on each half-code were physically linked on the plasmid in the previous round of cloning, we can link the phenotype expressed by the fluorescence to the leucine zipper.
The full MiCode assembly, done in a single-pot reaction to combine the 40 bait and 40 prey zippers into a 1,600 member library.
