• Recombi nation-mediated genetic engi neering • λ -Red recombination system – Gam – Exo – Beta DNA replication fork Costantino & Court. PNAS (2003)
Multiplex Automated Genome Engineering Expediting the design & evolution of organisms with new & improved properties • Small-scale genome engineering • Simultaneously targets many genetic locations at high efficiency (>30%) • Combinatorial genomic diversity across whole cell populations (10 9 ) Nature , 7257:894-8 (2009) Science , 333:348-53 (2011)
New genetic codes Pathway engineering Wang & Isaacs et al, Nature 2009 Isaacs et al., Science. 2011; 333(6040), 348-353.
Design challenges Challenge 1 ? How can we predict the outcome? SOLUTION: Computer modeling of multiplex recombination
1 2 3 • We assume the oligos bind + – only completely ( p 1 , r 1 = r 1 ) 1 ( p 2 , r 2 = 3 r 1 ) 2 – only intended targets ( p 3 , r 3 = 2 r 1 ) 3 – at an empirically estimated efficiency in E. coli (Wang & Isaacs et al, Nature 2009; Pr(6 r 1 ) = Pr( r 1 + r 2 + r 3 ) = p 1 p 2 p 3 Isaacs et al, Science 2011) 1 2 3 1 2 3 • Our algorithm solves + – the NP-complete subset sum Pr(5 r 1 ) = Pr( r 2 + r 3 ) = (1 - p 1 ) p 2 p 3 2 3 problem in time O(2 n /2 ) 1 2 3 (Horowitz & Sahni, 1974) + – a degenerate case in Pr(4 r 1 ) = Pr( r 1 + r 2 ) = p 1 p 2 (1 – p 3 ) polynomial time 1 2 (Wadyicki, Shah et al, 1973) 1 2 3 +
How the number of mutations per cell How each mutation accumulates accumulates over cycles over cycles How the minimum number of mutations How many off-target binding events might per cell increases over cycles happen, and how spontaneously
Technical challenges Challenge 2 Challenge 3 Challenge 4 Mutation Efficiency per Cycle 3h Electroporation Complex oligo pools Oligo breakdown MAGE efficiency = rate of oligo incorporation • concentration of oligo over time SOLUTION: MAGE in naturally competent organisms Science , 333:348-53 (2011)
• Natural competence A. baylyi – Cells actively take up/incorporate DNA from their environment • Genetic diversity Genescope, 2010 • Nucleotide recycling B. subtilis • HR-Mediated DNA repair • Advantages – Eliminate electroporation – Increase diversity of MAGE- able organisms
1. Identify suitable host organism 2. Knock out mismatch repair (MMR) 3. Construct a MAGE assay – Construct a universal cassette to quantify genetic modifications 4. Construct a library of ssDNA- binding recombinases 5. Design mutagenic oligos
Acinetobacter baylyi ADP1 (Gram -) – Close cousin of E. coli – High degree of natural competence – Useful natural pathways • Strains acquired: – A. baylyi ADP1 WT (Genoscope) – ADP1 Δ mutS (Genoscope) Genescope, 2010 Bacillus subtilis (Gram +) – Prototype for all Gram-positive organisms – Industrially relevant pathways • Strains acquired: – B. subtilis 168 (Bacillus Genetic Stock Center) – Bs PY79 (BGSC) – Bs 1A833 (BGSC)
• Why Δ mutS mutants? – MMR deficient – Increased incorporation of mutagenic oligos E. coli strains lacking mismatch repair genes ( mutH, mutL, mutS) Improved recombination efficiency: 10 -6 -10 -4 0.10-0.15 (> 1000 fold increase!)
1. Identify suitable host organism 2. Knock out mismatch repair (MMR) 3. Construct a MAGE assay – Construct a universal cassette to quantify genetic modifications 4. Construct a library of ssDNA- binding recombinases 5. Design mutagenic oligos
• We needed something to test that will give a clear change in phenotype , regardless of transformation/recombination efficiency or organism genotype • If designed rationally, such a cassette could be widely applied for new organisms • What parts do we need? • How do we make it universal? • How do we make it?
Universal Plasmid – pBAV1K-T5 (Bryksin and Matsumura, PLoS ONE, 2012) • Plasmid acquired from Addgene • Successful transformation in diverse bacterial species • High copy numbers in A. baylyi, B. subtilis, and E. coli • BioBrick-compatible Negative control • We were able to show efficient B. subtilis 168 transformation in all three of + pBAV1K our organisms
1. Promoter – T5 promoter element (from pBAV1K-T5) 2. Reporter – LacZ reporter gene (from E. coli genome) 3. Selectable marker (defective) – Defective Chloramphenicol resistance (nonsense mutation, from E. coli EcFI5) 4. Selectable marker (active) – Tetracycline resistance (from pBR322) 5. Terminator – T1 terminator (from pBAV1K-T5)
Cross-over PCR • Relies on homology between parts 1. Generate homology through PCR amplification 2. Mix templates, amplify with end primers • Second round to complete promoters
• Cassette successfully amplified (white arrow) • To verify function: – Cloned into pBAV1K using BioBrick restriction sites – Transformed into E. coli and A. baylyi by electroporation, and A. baylyi using natural competence
Negative Positive Control Control (pBAV1K) E. coli MachI ( Δ lacZ ) on Kan – X-Gal – IPTG plates • Blue colonies on all experimental transformations
Kan Kan Kan Kan Kan Kan + Cm + Tet + Cm + Tet Growth: MachI untransformed MachI transformed
Negative Positive Control Control (pBAV1K) A. baylyi ADP1 on Kan – X-Gal – IPTG plates • Electroporation and natural transformation
1. Identify suitable host organism 2. Knock out mismatch repair (MMR) 3. Construct a MAGE assay – Construct a universal cassette to quantify genetic modifications 4. Construct a library of ssDNA- binding recombinases 5. Design mutagenic oligos
Integrated Origin • We don’t know which enzyme Recombinase will work best for each species Gp35 Phage SPP1, B. subtilis Gp61 Phage Che9c, M. smegmantis – Test homologs isolated from orf48 Phage A118, L. monocytogenes diverse species orf245 Phage u136.2, L. lactis – Use bioinformatic techniques to EF2132 Enterococcus faecalis identify putative recombinases s065 SXT element, V. cholerae Plu2935 Photorhabdus luminescens orfC Legionella pneumophila Gp20 Phage phiNM3 , S. aureus recT Rac prophage, E. coli bet Phage lambda, E. coli EF2132-exo Enterococcus faecalis-exo s065-exo SXT element, V. cholerae-exo orfC-exo Legionella pneumophila-exo recT-exo Rac prophage, E. coli-exo bet-exo Phage lambda, E. coli-exo Datta et al., PNAS (2008)
• To each homolog isolated, we added the T5 promoter (pBAV1K) and XbaI and SpeI sites • We cloned into pBAV1K and sequenced : • bet • recT • plu2935 • orfC • s065-exo • recT-exo • Other homologs in progress • Re-cloned into pSB1C3 (BioBrick) – bet - BBa_K810000
1. Identify suitable host organism 2. Knock out mismatch repair (MMR) 3. Construct a MAGE assay – Construct a universal cassette to quantify genetic modifications 4. Construct a library of ssDNA- binding recombinases 5. Design mutagenic oligos
Test Oligo Optimal Conditions MAGE in naturally competent organisms
Spencer Katz David Lim Special thanks to Scott Strobel and Christopher Incarvito for lab space on Yale’s West Campus. Farren Isaacs, Advisor Assistant Professor of Molecular, Cellular, and Aaron Lewis Andriana Lebid Developmental Biology, Yale University Graduate advisors • Natalie Ma, Isaacs lab • Ryan Gallagher, Isaacs lab • Edward Barbieri, Isaacs lab • Alexis Rovner, Isaacs lab • Darryl Reeves, Isaacs lab • Jen Nguyen, Modis lab • Dan Spakowicz, Strobel lab Aaron Hakim Jason No
• Altman and Breaker labs, for bacterial strains • CAD/Art Services, for transparencies • Geneious, for software licensing • Jiming Wang and Wuyi Meng • Kappa Biosystems, for PCR reagents • Kaury Kucera, for primer design advice • Lloyd Chen, for laboratory assistance • New England Biolabs, for material support • Roeder and Grindley labs, for material support • Professor Clay, for material support • Professor Schatz & Liz Corbett for a sonicating water bath and thermocycler • Raymond and Beverly Sackler Institute • William Segraves and the Yale College Dean's Office, for student fellowships • Yale Science and Engineering Association • Yale University Department of Physics • Yale University Department of Molecular, Cellular, and Developmental Biology • Yale University Department of Molecular Biophysics & Biochemistry • Yale University West Campus
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