Talk:CH391L/S12/MAGE lycopene production, CAGE "Amberless" E. coli

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(New page: *'''~~~~''':There's an NAR paper about using modified bases in the oligos to avoid having to knock out MMR repair to get high efficiency incorporation. Can you summarize? <cite>Wang2011</c...)
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*'''[[User:Jeffrey E. Barrick|Jeffrey E. Barrick]] 13:13, 14 April 2012 (EDT)''':There's an NAR paper about using modified bases in the oligos to avoid having to knock out MMR repair to get high efficiency incorporation. Can you summarize? <cite>Wang2011</cite>.  
*'''[[User:Jeffrey E. Barrick|Jeffrey E. Barrick]] 13:13, 14 April 2012 (EDT)''':There's an NAR paper about using modified bases in the oligos to avoid having to knock out MMR repair to get high efficiency incorporation. Can you summarize? <cite>Wang2011</cite>.  
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*'''[[User:Jeffrey E. Barrick|Jeffrey E. Barrick]] 13:22, 14 April 2012 (EDT)''': The MAGE papers cite beta as being the important activity, but also that the mechanism is not entirely understood. All of the strains used seem to have all three of the Lambda Red proteins (alpha, beta, gamma) integrated into their chromosomes in a way that they are induced at the same time? Here are some relevant strain construction details.
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<blockquote>In brief, a defective Phage λ-Red construct was introduced by P1 transduction into E. coli MG1655 at the bioA location to produce EcNR1 (ΔbioA::λ-Red-bla). The relevant λ genes Redα, Redβ and Redγ are under regulation of the pL promoter and the temperature sensitive cI857 repressor. EcNR2 was made by using λ-Red homologous recombination to replace mutS with a chloramphenicol acetyltransferase (cat) cassette in EcNR1, thereby generating the ΔmutS::cat genotype. EcZS2 was made by introducing a kanamycin resistance (kan) cassette to replace the recA gene in EcNR1, generating a ΔrecA::kan genotype."<cite>Wang2011</cite></blockquote>
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*'''[[User:Jeffrey E. Barrick|Jeffrey E. Barrick]] 13:22, 14 April 2012 (EDT)''':What's the relationship of MAGE to "recombineering"?
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Revision as of 12:22, 14 April 2012

  • Jeffrey E. Barrick 13:13, 14 April 2012 (EDT):There's an NAR paper about using modified bases in the oligos to avoid having to knock out MMR repair to get high efficiency incorporation. Can you summarize? [1].
  • Jeffrey E. Barrick 13:22, 14 April 2012 (EDT): The MAGE papers cite beta as being the important activity, but also that the mechanism is not entirely understood. All of the strains used seem to have all three of the Lambda Red proteins (alpha, beta, gamma) integrated into their chromosomes in a way that they are induced at the same time? Here are some relevant strain construction details.
In brief, a defective Phage λ-Red construct was introduced by P1 transduction into E. coli MG1655 at the bioA location to produce EcNR1 (ΔbioA::λ-Red-bla). The relevant λ genes Redα, Redβ and Redγ are under regulation of the pL promoter and the temperature sensitive cI857 repressor. EcNR2 was made by using λ-Red homologous recombination to replace mutS with a chloramphenicol acetyltransferase (cat) cassette in EcNR1, thereby generating the ΔmutS::cat genotype. EcZS2 was made by introducing a kanamycin resistance (kan) cassette to replace the recA gene in EcNR1, generating a ΔrecA::kan genotype."[1]
  • Jeffrey E. Barrick 13:22, 14 April 2012 (EDT):What's the relationship of MAGE to "recombineering"?
  1. Wang HH, Xu G, Vonner AJ, and Church G. . pmid:21609953. PubMed HubMed [Wang2011]
    Modified bases enable high-efficiency oligonucleotide-mediated allelic replacement via mismatch repair evasion.

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