programming cells by multiplex genome engineering and ...€¦ · multiplex genome engineering and...
TRANSCRIPT
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Programming Cells by Multiplex Genome
Engineering and Accelerated Evolution
By Harris H. Wang, Farren J. Issacs, Peter A. Carr, Zachary Z. Sun, George Xu, craig R.
Forest & George M. Church.
Presented by Alie Doolittle for 20.385 4.1.2010
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Hypothesis
• Multiplex Automated Genome Engineering (MAGE) allows parallel and continuous accelerated evolution – Designed to target many cellular locations
on chromosomes across a population of cells
– Cyclical and scalable
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Methods in Directed Evolution Currently:
Gene mutation → Selection →Amplification PCR DNA Shuffling
(Sexual PCR)
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MAGE Directed Evolution
Allows for Rapid and Continuous Generation of Genetic Changes
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MAGE - Multiplex Automated Genome Engineering
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MAGE Efficiency Over 30% genetic modification per cell
every 2-2.5 hours
Mismatch and Insertion Efficiency
Amount of homologous Sequence
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MAGE Efficiency
∆G between Oligo and Deletion Probability Chromosome Sequence
Size of the Deletion Replacement Efficiency
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Sequence Diversity over Multiple MAGE cycles
Increased divergence from wild type demonstrated with increased MAGE cycles
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MAGE Diversity • Degree of Sequence Variation:
– The number of loci – The number of MAGE cycles
• The frequency locus modification can be demonstrated through a binomial distribution:
M - Mutation Rate
N - MAGE Cycles
K - Targeted Loci
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Automated MAGE
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Ex. Lycopene Production
DXP Biosynthesis Pathway: - 20 Genes known to increase production (RED) - 4 genes known to inhibit production (BLUE)
Total: 15 billion variants
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Ex. Lycopene Production
Red - Lycopene production White - Little Production
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Ex. Lycopene Production
1. Characterize isolate mutants to to asses different tuning pathways.
2. Observe the effect of multiple mutations.
Can use MAGE to specifically target specific genes through the use of oligos with defined sequence
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MAGE Possibilities
• Modify and Sample Different Strength RBSs.
• Inactivate Protein Coding Sequences. • Enhance incorporation of non native
amino acids. – Construct safer multi-virus strains.
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Looking Ahead
• Ethically how will the MAGE technique be used in higher organisms and medicine?
• Could this create a highly mutating strain? What if this escaped into the wild?
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Complete Chemical Synthesis, assembly, and Cloning of a Mycoplasma
genitalium Genome By Gibson DG, Benders GA, Andrews-
Pfannkoch C, Denisova EA, Baden-Tillson H, Zaveri J, Stockwell TB, Brownley A, Thomas
DW, Algire MA, Merryman C, Young L, Noskov VN, Glass JI, Venter JC, Hutchison
CA 3rd, Smith HO.
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Genome Assembly
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Confirmation of 580kb Genome
through CHEF Gel Analysis.