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

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Current revision (18:18, 16 April 2012) (view source)
 
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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:James L. Bachman|James L. Bachman]] 12:44, 16 April 2012 (EDT)''':To avoid the MMR of ''L. reuteri'' the authors designed oligos with mismatch pairs to target the ''rpoB'' gene, which they had predicted the amino acid change would result in the rifampicin-resistant phenotype. Mutations such as C·C mismatch, which had been shown to be not efficiently recognized by ''E. Coli'' MMR were created in the oligos. MMR avoidance efficiency was determined by comparing rate of recombination of MMR deficient strains (mutS1- and mutL-) with the wild-type strains. The oligo completely avoids MMR if it has a similar number of recombinations in wild type as it does in MMR deficient strains. The only oligo to completely avoid MMR was that with five consecutive mismatch mutations. The recombination frequency was high enough such that authors were able to show that targeted "non-selected recombineering" could be performed by using oligos that avoid MMR.<cite>Wang2011</cite> [[Image:L.reuteri.gif|thumb|200px|<cite>Wang2011</cite>]]
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**'''[[User:James L. Bachman|James L. Bachman]] 12:44, 16 April 2012 (EDT)''':To avoid the MMR of ''L. reuteri'' the authors designed oligos with mismatch pairs to target the ''rpoB'' gene, which they had predicted the amino acid change would result in the rifampicin-resistant phenotype. Mutations such as C·C mismatch, which had been shown to be not efficiently recognized by ''E. Coli'' MMR were created in the oligos. MMR avoidance efficiency was determined by comparing rate of recombination of MMR deficient strains (mutS1- and mutL-) with the wild-type strains. The oligo completely avoids MMR if it has a similar number of recombinations in wild type as it does in MMR deficient strains. The only oligo to completely avoid MMR was that with five consecutive mismatch mutations. The recombination frequency was high enough such that authors were able to show that targeted "non-selected recombineering" could be performed by using oligos that avoid MMR.<cite>lactic2012</cite> [[Image:L.reuteri.gif|thumb|200px|<cite>lactic2012</cite>]]
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***'''[[User:James L. Bachman|James L. Bachman]] 18:18, 16 April 2012 (EDT)''':Edit: On the webpage for the Church paper you linked, I mistakenly clicked on the wrong "Full HTML" link on the Oxford Journals webpage... To summarize the Church group's experiment: 90-mer oligos were designed that contained mismatch mutations such as C·C, which are inefficiently corrected by ''E. coli'' MMR machinery. The oligos contained ribose or nucleobase chemical modifications and screened using lacZ and malK genes. In a MMR capable strain, it was shown that 2′-Fluoro-deoxyUridine, 5-Methyl-deoxyCytidine, 2,6-Diaminopurine or Iso-deoxyGuanosine containing oligos increased recombination frequency of the mismatch by 10-20 fold over the non-modified oligos. While Oligos with phosphorothioated bases A, C and T at the mismatch site had an average of 5-fold improvement over non-phosphorothioated oligos. They extended this further by taking the best two chemically-altered bases for recombination, 2′-Fluoro-deoxyUridine or 5-Methyl-deoxyCytidine, and showed that if these were used to incorporated a mismatch commonly fixed by MMR machinery such as G:T or C:T, that the MMR efficiency was nearly equal to that for a wild-type inefficiently fixed C:C mismatch.<cite>Wang2011</cite>
*'''[[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.
*'''[[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.
<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>
<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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#Isaacs2011 pmid=21764749
#Isaacs2011 pmid=21764749
#Costantino2010  pmid=14673109
#Costantino2010  pmid=14673109
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#lactic2012 pmid=22328729
</biblio>
</biblio>

Current revision

  • 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].
    • James L. Bachman 12:44, 16 April 2012 (EDT):To avoid the MMR of L. reuteri the authors designed oligos with mismatch pairs to target the rpoB gene, which they had predicted the amino acid change would result in the rifampicin-resistant phenotype. Mutations such as C·C mismatch, which had been shown to be not efficiently recognized by E. Coli MMR were created in the oligos. MMR avoidance efficiency was determined by comparing rate of recombination of MMR deficient strains (mutS1- and mutL-) with the wild-type strains. The oligo completely avoids MMR if it has a similar number of recombinations in wild type as it does in MMR deficient strains. The only oligo to completely avoid MMR was that with five consecutive mismatch mutations. The recombination frequency was high enough such that authors were able to show that targeted "non-selected recombineering" could be performed by using oligos that avoid MMR.[2]
      [2]
      [2]
      • James L. Bachman 18:18, 16 April 2012 (EDT):Edit: On the webpage for the Church paper you linked, I mistakenly clicked on the wrong "Full HTML" link on the Oxford Journals webpage... To summarize the Church group's experiment: 90-mer oligos were designed that contained mismatch mutations such as C·C, which are inefficiently corrected by E. coli MMR machinery. The oligos contained ribose or nucleobase chemical modifications and screened using lacZ and malK genes. In a MMR capable strain, it was shown that 2′-Fluoro-deoxyUridine, 5-Methyl-deoxyCytidine, 2,6-Diaminopurine or Iso-deoxyGuanosine containing oligos increased recombination frequency of the mismatch by 10-20 fold over the non-modified oligos. While Oligos with phosphorothioated bases A, C and T at the mismatch site had an average of 5-fold improvement over non-phosphorothioated oligos. They extended this further by taking the best two chemically-altered bases for recombination, 2′-Fluoro-deoxyUridine or 5-Methyl-deoxyCytidine, and showed that if these were used to incorporated a mismatch commonly fixed by MMR machinery such as G:T or C:T, that the MMR efficiency was nearly equal to that for a wild-type inefficiently fixed C:C mismatch.[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]
  • James L. Bachman 14:14, 16 April 2012 (EDT): In this paper by Constantin et al.[3], it was shown that only the beta protein is involved in ssDNA recombination, but even then, alpha and gamma proteins were deleted. Since the beta protein incorporates the ssDNA through an annealing and replication-dependent mechanism rather than homologous recombination [1], it could be that having the other proteins activated, it may not have a negative effective with ssDNA, but I have not yet found a paper verifying this assumption.
  • Jeffrey E. Barrick 13:22, 14 April 2012 (EDT):What's the relationship of MAGE to "recombineering"?
    • James L. Bachman 11:04, 16 April 2012 (EDT):It seems that recombineering can be defined as "Engineering recombinant DNA molecules by in vivo homologous recombination".[1] The technique is based on the λ-Red system for either dsDNA or ssDNA incorporation. Since MAGE is an automated system that uses the λ-Red ssDNA incorporation as a means to generate recombination across the whole genome, it should be considered automated recombineering.
  • Jeffrey E. Barrick 13:24, 14 April 2012 (EDT):"Okazaki" is capitalized (it's someone's name). Please italicize E. coli.
  • Jeffrey E. Barrick 13:26, 14 April 2012 (EDT):Please summarize some of our discussion during class about how many cells die at each electroporation cycle, how much regrowth occurs between cycles, and the fact that apparently some portion of the population becomes "resistant" to MAGE.
    • James L. Bachman 10:29, 16 April 2012 (EDT):Through automating the MAGE process, the Church group was able to get MAGE cycles down to 2-2.5hr. At the electroporation step a high voltage of 18kV/cm was used, killing 95% of the cells in that cycle, which is then recovered to mid-log phase at a concentration of 7x10^8 cells/ml in liquid media for the next cycle. As for the MAGE "resistant" populations, I think this was in context of how well the cells incorporated the stop codon mutations at the targeted position versus no incorporation. All the CAGE paper mentions of the 42% of cell populations remaining unchanged is "[this] measurement suggests the evolution of two types of cells in our mixed cultures: one that appears largely resistant to allelic replacements and, and another that readily permits them".[4] Other discussion involved efficiency of targeted mutations versus background mutations. Isaacs et al. said the efficiency of MAGE only allowed for 6-8 cell divisions per cycle, which lowered background mutations. To analyze these secondary mutations, the authors performed BLAST alignments of the oligos against the entire genome. Finding that ~270 oligos had some homology to other sequences and that this corresponded to decreased recombination frequency. Of the mutations outside the targeted 90-bp regions were: 6 substitutions, 0 insertions, and 3 deletions, contrasted to targeted regions: 4 substitutions, 1 insertion, 28 deletions. [4]
  • Jeffrey E. Barrick 13:29, 14 April 2012 (EDT):Link to unnatural amino acid incorporation and genome synthesis topics?
    • James L. Bachman 10:29, 16 April 2012 (EDT):Added both, to note, whole-genome synthesis was actually talked about in the conclusion to the church MAGE paper.

References

  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.

  2. van Pijkeren JP and Britton RA. . pmid:22328729. PubMed HubMed [lactic2012]
  3. Isaacs FJ, Carr PA, Wang HH, Lajoie MJ, Sterling B, Kraal L, Tolonen AC, Gianoulis TA, Goodman DB, Reppas NB, Emig CJ, Bang D, Hwang SJ, Jewett MC, Jacobson JM, and Church GM. . pmid:21764749. PubMed HubMed [Isaacs2011]
  4. Costantino N and Court DL. . pmid:14673109. PubMed HubMed [Costantino2010]
All Medline abstracts: PubMed HubMed
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