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Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement

Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement. Isaacs, Farren J. et al. Science , Vol 333, 2011. Presented by: PJ Velez. Useful Definitions. Conjugative Assembly genome engineering (CAGE) Method - Large Scale Engineering

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Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement

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  1. Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement Isaacs, Farren J. et al. Science, Vol 333, 2011 Presented by: PJ Velez

  2. Useful Definitions • Conjugative Assembly genome engineering (CAGE) Method - Large Scale Engineering • Multiplex Automated Genome Engineering (MAGE) Method – Small Scale Engineering • λ Red Protein- Proteins that promote recombination and are mutagenic. • RF1/RF2 – Release factors in E. Coli that recognize stop codons

  3. Goals and Motivation • Long Term Goal: Successfully modify genetic code • Long Term Impact: • Novel Biological System Properties • Easier incorporation of unnatural Amino Acids • Short Term Approach: • Replace all TAG stop codons with TAA in viable E. Coli • RF1 deletion mutant still viable

  4. Overall Strategy

  5. MAGE • Genome split into 32 regions of <10 genes with TAA codon • 18 MAGE Cycles • Assays to identify greatest number of codon conversion and measure frequencies

  6. MAGE • Some Cells more susceptible to mutations • Top clone found for each region • Also looked at potential unintended mutations • BLAST • Sanger Sequencing

  7. CAGE • 32 clones put into pairs • Donor Strand • oriT-kan and positive selectable marker • Recipient Strand • Positive-Negative selectable marker and different positive marker

  8. Final Experiments • Performed genome sequencing on two dysfunctional strains and a control after MAGE/CAGE • 1 error per genome per 9 replications • Hypergeometric distribution • Determine enrichment level across three strains • Problem: No figures or data shown in paper • Buried in 90 pages of supplement

  9. Conclusions and Future Directions • Successfully replaced all TAG occurrences with TAA codons • Improve future genome engineering efforts • Dynamic method to introduce change in cell • Help refine existing genome annotation • Already been cited four times • Came out last July

  10. Supplementary Slides for Discussion

  11. Issues with Final Figure • Overall layout not really specified • Media? • Order of Rows? • Yellow Arrows?

  12. Other points for Discussion • Successfully show what they set to? • Final Experiments worthy of being published?

  13. MAGE • Sanger Sequencing verified presence of conversion and secondary mutations • Look at 300 bp surrounding replaced site

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