Talk:CH391L/S12/GeneandGenomeSynthesis

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*'''[[User:Michael Hammerling|Michael Hammerling]] 14:50, 15 February 2012 (EST)''': What types of sequences are particularly difficult to synthesize using these methods?
*'''[[User:Michael Hammerling|Michael Hammerling]] 14:50, 15 February 2012 (EST)''': What types of sequences are particularly difficult to synthesize using these methods?
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::*'''[[User:Joe Hanson|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)
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::'''[[User:Joe Hanson|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)
*'''[[User:Peter Otoupal|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?
*'''[[User:Peter Otoupal|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?

Revision as of 16:40, 18 February 2012


  • 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].
  • 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?
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