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Team:Queens Canada/Guide/DNA
From 2012.igem.org
Control
ChimeriQ - Description
Cut Sites
Cloning Methods
Cloning Method
When cloning parts into our construct, we chose to use PCR overlap extension and digestion/ligation
techniques. In order to perform PCR Overlap extension (our preferred method of cloning) we were
required to fabricate a set of primers that were ligated onto the insert. These primer extensions served
to add overlap sites that match those of the parts we were adding. This allowed for PCR to extend
the part onto another part with a matching overlap site; with the primers on either end of the inserts
acting as the site of initiation for Taq polymerase. Our primary resource was this paper. A picture
representation of PCR-Overlap Extension can be seen below:
The other method of cloning made use of the cut sites found in our insert and a few restriction enzymes.
Early on we noted the BioBrick standard caused a very problematic frame-shifts within the scar when
ligating 2 or more parts. We tried to circumvent the problem by adding 1 or 2 nucleotides onto our DNA
sequence to fix the frame but found this to be tedious due to the fact that this varied based on the part
we wanted to ligate.
Therefore we started doing research into other assembly standards to see if any of them alleviated
the problem. We judged the merits and flaws of each one (a list of the assembly standards considered
can be found here). What we searched for was a standard that would avoid frame-shifts and nonsense
mutations when doing protein fusion, avoid N-terminal destabilization signals and dam methylation sites
and preserve most of the restriction enzymes from the original BBa standard.
Considering these reasons, we chose to switch our prefixes and suffixes to Tom Knight’s BBa 2 standard
that allowed seamless in-frame ligations between multiple sequences of successive DNA. This change
resulted in only 1 restriction enzyme (Xba1 to Nhe1) that differed from original BBa standard therefore
providing the flexibility we needed to simultaneously work with both standards. For ligation, we used T4
ligase. A pictorial representation of the BBa 2 standard can be seen below:
Prefix
5' GAATTC...ACTAGT
GCTAGC...CTGCAG 3'
EcoRI
SpeI ...part... NheI
PstI
Fusing two parts would then leave the following scar:
5' ...part A... A
We also considered making our own artificial restriction enzymes in order to cut DNA off-site. To do
so, we would have made zinc fingers to bind DNA and combined them with enzymes capable of cutting
away from the binding site. This would allow us to cut as close to the gene as possible.
Suffix
GCTAGT
S ...part B... 3'
Parts for protein expression
CohDoc
