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Team:Queens Canada/Guide/DNA
From 2012.igem.org
DNA
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 Suffix
EcoRI SpeI NheI PstI
5' GAATTC...ACTAGT ...part... GCTAGC...CTGCAG 3'
Fusing two parts would then leave the following scar:
5' ...part A... GCTAGT ...part B... 3'
A S
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.
When designing our flagella construct we examined a number of different ways of creating fusion proteins. Using the original BioBrick assembly standard does not work for fusion proteins, as it results in a frame shift and stop codon in the scar. At first we worked on creating a new method of fusion protein production, however we ran into many of the same problems as the standards that were already present. We went through the various fusion standards present on the openwetware web page, analyzing their different merits, eventually settling on Tom Knight’s BB-2 standard. You can follow this link for some information on the various standards we examined.
Biofusion (Silver Lab)
Prefix Suffix
5’ GAATTC GCGGCCGC T TCTAGA ...part... ACTAGT A GCGGCCG CTGCAG 3’
EcoRI NotI XbaI SpeI NotI PstI
This results in the following scar:
5’ ...part A...ACTAGA...part B...3’
ThrArg
The Biofusion standard slightly adjusted the original BioBrick standard such that the scar would now code for arginine instead of a stop codon, and the fusion would be in frame. Also, this standard uses the same enzymes as the original, so no new enzymes must be purchased. However, some issues do arise.These include the fact that the arginine present in the scar may be problematic in some cases due to its large size and the fact that it is coded by a rare codon. Also, Dam methylation may block cloning if the fused protein begins with Serine.
Fusion Parts (Freiburg iGEM 2007)
Prefix Suffix 5' GAATTC GCGGCCGC T TCTAGA TG GCCGGC...part...ACCGGT TAAT ACTAGT A GCGGCCG CTGCAG 3' EcoRI NotI XbaI Met NgoMI AgeI SpeI NotI PstI This results in the following scar: 5’ ...part A...ACCGGC...part B... 3’ ThrGly
The Fusion Parts standard proposed by Freiburg serves to allow fusion proteins but remove some of the disadvantages of the Biofusion standard. It does this by adding two new restriction enzymes: NgoMI and AgeI. This standard allows a less disruptive scar of threonine and glycine, while maintaining in frame fusions. However, this does require the purchase of two new restriction enzymes.
BglBricks (Berkeley)
Prefix Suffix 5’ GAATTC ATG AGATCT...part...GGATCC TAA CTCGAG 3’ EcoRI BglII BamHI XhoI This results in the following scar: 5’ ...part A...GGATCT...part B...3’ GlySer
The BglBricks standard allows in frame production of fusion proteins with a benign scar of of glycine and serine. The prefix and suffix are also quite simple, containing only the 2 restriction sites connected by 3 bases. However, this method uses 3 new enzymes: BglII, BamHI and XhoI, of which BglII and BamHI cannot be heat-inactivated.
BB-2 (Tom Knight)
Prefix Suffix
5’ GAATTC...ACTAGT...part...GCTAGC...CTGCAG 3’
EcoRI SpeI NheI PstI
This results in the following scar:
5’...part A...GCTAGT...part B... 3’
AlaSer
The BB-2 standard proposed by Tom Knight was the method we ended up following to produce our chimeric flagella. This standard requires the addition of only one new restriction enzyme (NheI), and produces in frame fusions with a benign scar of alanine and serine. Also, the NheI cut site is fairly rare in E. coli, decreasing the likelihood of having cut sites within proteins, and this enzyme can be heat-inactivated. The main concern with this method is that it has not yet been tested extensively to prove its value as a standard method of Biobrick assembly.
