Talk:CH391L/S12/GeneandGenomeSynthesis

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::'''[[User:Joe Hanson|Joe Hanson]] 15:26, 15 February 2012 (EST)''': Judging from the references in the end of their manual for the [http://tools.invitrogen.com/content/sfs/manuals/geneart_seamless_cloning_and_assembly_man.pdf 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 [http://en.wikipedia.org/wiki/Recombineering recombineering] methods. For those who care to look it up, in the tube, you probably get a mix of [http://www.ncbi.nlm.nih.gov/pubmed/9500923 λ phage Redβ protein], ''E. coli'' [http://www.ncbi.nlm.nih.gov/pubmed/9771703 RecE/RecT proteins] and [http://www.ncbi.nlm.nih.gov/pubmed/11118378 vaccinia virus DNA polymerase].
::'''[[User:Joe Hanson|Joe Hanson]] 15:26, 15 February 2012 (EST)''': Judging from the references in the end of their manual for the [http://tools.invitrogen.com/content/sfs/manuals/geneart_seamless_cloning_and_assembly_man.pdf 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 [http://en.wikipedia.org/wiki/Recombineering recombineering] methods. For those who care to look it up, in the tube, you probably get a mix of [http://www.ncbi.nlm.nih.gov/pubmed/9500923 λ phage Redβ protein], ''E. coli'' [http://www.ncbi.nlm.nih.gov/pubmed/9771703 RecE/RecT proteins] and [http://www.ncbi.nlm.nih.gov/pubmed/11118378 vaccinia virus DNA polymerase].
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::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). 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. All the first steps are at room temperature.  
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::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. [http://www.genebridges.com/gb/red_et_principles.php This website] has a cartoon that sort of lays out the process.  
*'''[[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?

Revision as of 16:32, 15 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.
  • Michael Hammerling 14:50, 15 February 2012 (EST): What types of sequences are particularly difficult to synthesize using these methods?
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