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Revision as of 11:13, 20 February 2012 by Joe Hanson (Talk | contribs)
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  • Jeffrey E. Barrick 11:17, 14 February 2012 (EST):You mentioned the Gene Art kits from Invitrogen in class. They advertise a lot of applications for stitching together up to ten fragments at a time. I guess no one really knows what's in the kits though?
Joe Hanson 15:26, 15 February 2012 (EST): Judging from the references in the end of their manual for the GeneArt Seamless Assembly Kit, I can gather that it's an in vitro recombination system derived from a some or all parts of a few recombineering methods. For those who care to look it up, in the tube, you probably get a mix of λ phage Redβ protein, E. coli RecE/RecT proteins and vaccinia virus DNA polymerase.
Again, this is just my guess, but the RecE/RecT, λ Redβ and vaccinia DNA Pol all work to take small regions of homology, and strand displace them into another duplex (sometimes using exonuclease activity). So if A shares 20bl with B, A and B would be combined into a new duplex with a nick. Make one of those pieces a plasmid backbone, and you end up getting a noncovalently bonded circular plasmid in the end. This is then transformed into recA E. coli strains and is repaired into a complete plasmid. This website has a cartoon that sort of lays out the process.
--Erik Quandt 15:51, 16 February 2012 (EST): This cloning method seems very similar to the "Infusion" kit from Clontech [1]. Although they don't explicitly tell you what enzyme they use like the GeneArt kit, I assume it is a just proprietary thermostable polymerase with exonuclease activity. The exonuclease chews back on a single strand to create overlapping homologies, annealing occurs, and then the polymerase activity kicks in to fill the missing bases. Since there is no ligase in the mix (unlike Gibson), nicks are left but are repaired by the cell once transformed. Here is a good paper describing the adaptation of this system for "Biobricking" [2].
  • Jeffrey E. Barrick 21:08, 18 February 2012 (EST): I overlapped with Sean Sleight when he was in the Lenski lab. I've been working on him to visit Austin sometime. Which of these many choices of techniques should the iGEM team be using for most constructs? What is the best commercial system versus the best homebrew? We need to build a pro/con table of techniques and which methods have advantages for certain applications as a class? Or I imagine it could be something like a dichotomous taxonomic key where you answer yes/no questions and it leads you to the technique you should use. Example That might be a cute feature for an iGEM team website that adds to the community. Or does one already exist?
  • Michael Hammerling 14:50, 15 February 2012 (EST): What types of sequences are particularly difficult to synthesize using these methods?
Joe Hanson 15:40, 18 February 2012 (EST)Repetitive sequences are difficult to synthesize by any of these methods because they can recombine and mis-anneal, either introducing new repeats or deleting sequences. Repetitive sequences can also recombine in the hosts used for assembling larger synthetic genomes like yeast and E. coli. Also, if the sequence has too high a percentage of either AT or GC sequence, it can throw off the annealing protocols that are used to assemble the genes (not to mention that those oligos often have strange secondary structures)
  • Peter Otoupal 12:38, 17 February 2012 (EST):I'm still a little confused on the benefits of minimizing a genome. How does it benefit an organism to remove these extraneous, unused genes?
  • Jeffrey E. Barrick 09:19, 20 February 2012 (EST):Sometimes these genes are directly bad for the organism (like toxin-antitoxin gene pairs we we will talk about next) or prophage that can re-activate. Often, they are just a possible complication. The more parts you have, the more chances you have of some unintended consequence when you add a new part.
  • Joe Hanson 10:13, 20 February 2012 (EST): There's also just the basic biological observations that you'd get by determining the minimal set of genes to sustain life. In the precursor organism, you could argue that the genes maybe aren't "extraneous", there's clearly been natural selection to maintain what might look like "redundant" genes. Venter's original idea in making a minimal genome was to create kind of a basic chassis that parts could be added to to create customized microbes.
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