Multiplex DNA synthesis and some applications

1. Multiplex DNA synthesis and some applications Farren Isaacs June 22, 2005 ALife Boston Church Lab Department of Genetics Harvard Medical School

2. Genome Sequencing Technologies: “the framework” >ENST00000262479 [p53]GCAGCCAGACTGCCTTCCGGGTCACTGCCATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCCCTCTGAGTCAGGAAACATTTTCAGACCTATGGAAACTACTTCCTGAAAACAACGTTCTGTCCCCCTTGCCGTCCCAAGCAATGGATGATTTGATGCTGTCCCCGGACGATATTGAACAATGGTTCACTGAAGACCCAGGTCCAGATGAAGCTCCCAGAATGCCAGAGGCTGCTCCCCGCGTGGCCCCTGCACCAGCAGCTCCTACACCGGCGGCCCCTGCACCAGCCCCCTCCTGGCCCCTGTCATCTTCTGTCCCTTCCCAGAAAACCTACCAGGGCAGCTACGGTTTCCGTCTGGGCTTCTTGCATTCTGGGACAGCCAAGTCTGTGACTTGCACGTACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGGCCAAGACCTGCCCTGTGCAGCTGTGGGTTGATTCCACACCCCCGCCCGGCACCCGCGTCCGCGCCATGGCCATCTACAAGCAGTCACAGCACATGACGGAGGTTGTGAGGCGCTGCCCCCACCATGAGCGCTGCTCAGATAGCGATGGTCTGGCCCCTCCTCAGCATCTTATCCGAGTGGAAGGAAATTTGCGTGTGGAGTATTTGGATGACAGAAACACTTTTCGACATAGTGTGGTGGTGCCCTATGAGCCGCCTGAGGTTGGCTCTGACTGTACCACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAACCGGAGGCCCATCCTCACCATCATCACACTGGAAGACTCCAGTGGTAATCTACTGGGACGGAACAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTGGGAGAGACCGGCGCACAGAGGAAGAGAATCTCCGCAAGAAAGGGGAGCCTCACCACGAGCTGCCCCCAGGGAGCACTAAGCGAGCACTGCCCAACAACACCAGCTCCTCTCCCCAGCCAAAGAAGAAACCACTGGATGGAGAATATTTCACCCTTCAGATCCGTGGGCGTGAGCGCTTCGAGATGTTCCGAGAGCTGAATGAGGCCTTGGAACTCAAGGATGCCCAGGCTGGGAAGGAGCCAGGGGGGAGCAGGGCTCACTCCAGCCACCTGAAGTCCAAAAAGGGTCAGTCTACCTCCCGCCATAAAAAACTCATGTTCAAGACAGAAGGGCCTGACTCAGAC “The sequence provides the framework upon which all the genetics, biochemistry physiology, and ultimately phenotype depend. It provides the boundary for scientific inquiry. The sequence is only the first level of understanding the genome. All genes and control elements must be identified; their functions in concert as well as in isolation, defined; their sequence variation worldwide described; and the relation between genome variation and specific phenotypic characteristics determined. Now we know what we have to explain.” J.C. Venter et al. Science 291 (2001) Shendure J, Mitra R, Varma C, Church GM, 2004 Nature Reviews of Genetics

3. Sequencing Technologies Systems Biology Synthetic Biology Synthesis Technologies

4. Cellular Phone: Designed and built by engineers EVERY component is characterized Cellular Network: Exhibit remarkably robust, precise behavior in the absence of our understanding

5. Synthetic Biology • Construction of small gene networks from well-characterized biological parts, guided by models Toggle Switch Gardner, Cantor & Collins Nature 403 (2000) Repressilator Elowitz & Leibler Nature 403 (2000) Good Review: Hasty, McMillen & Collins Nature 420 (2002)

6. Synthetic Biology Engineered Riboregulators Isaacs et al. Nature Biotech 22 (2004) • Design of new biological parts Ligand-controlled Riboregulators Bayer & Smolke Nature Biotech 23 (2005)

7. Modular Cell Biology Modules: composed of many types of molecules - DNA, RNA, proteins, small molecules - which have discrete functions that arise from interactions among their components Hartwell, Hopfield, Leibler, Murray Nature 402, C46 (1999) Arnone & Davidson Development 124, 1851 (1997) Synthetic Biology  Systems Biology BiologicalComplexity • reduce the complexity of networks from natural complex biological setting to isolate and study modular components that perform a specific function Advanced Synthesis Technologies

8. Multiplex DNA Synthesis from Programmable Microchips Tian et al. Nature 432 (2004)

9. Int Xis TF4 1 Int Xis TF3 2 Int Xis TF5 3 Int Xis TF6 4

13. Int Xis TF4 1 Int Xis TF3 2 Int Xis TF5 3 Int Xis TF6 4

14. Int Xis TF4 1 Int Xis TF3 2 Int Xis TF5 3 Int Xis TF6 4 Cell Counter (IGEM Summer '04) Boston University • Will Blake • Jim Flanigon • Farren Isaacs • Ellen O’Shaughnessy • Neil Patel • Margot Schomp • Jim Collins Harvard University • John Aach • Patrik D'haeseleer • Gary Gao • Jinkuk Kim • Xiaoxia Lin • Nathan Walsh • George Church http://theory.med.harvard.edu/SynBio/

16. Phage Int/Xis system Phage attachment sites attP O P P’ O B B’ attB Bacterial attachment sites Int + Xis Int Integrated Left attachment sites attL Integrated Right attachment sites attR O O B P’ P B’ Stably integrated prophage

17. Why Integrases – Excisionases? • High fidelity – site specific recombination • Reversible – excision just as reliable as integration • Specific – each integrase recognizes its own att sites, but no others • Numerous – over 300 known Tyr integrases and ~30 known Ser integrases • Efficient – very few other factors needed to integrate or excise • Extensively used – Phage systems well-characterized and used extensively in genetic engineering (e.g., the GATEWAY cloning system by Invitrogen)

18. Int/Xis system with inverted att sites Phage attachment sites attP Bacterial attachment sites attB* 0 O O P P’ B B’ Int + Xis Int Integrated Right attachment site attR Integrated Left attachment site attL* 1 O O P P’ B B’

19. Int2 Int2 Xis2 Rpt1 int2 xis2 rpt1 int1 xis1 reporter2 attR2 – – attL2* term int2 xis2 reporter1 attR1–term– attL1* Int1 Xis1 Rpt2 Int1 int1 xis1 rpt2 int1 int1 xis1 reporter2 int2 xis2 reporter1 attP2–term–attB2* attP1– – attB1* term Full Cycle of Two ½-bits int2 int2 xis2 reporter1 1 attR1–term– attL1* int1 xis1 reporter2 2 attP2–term– attB2*

20. Design Composite half bits in BioBricks Two 2kb composite parts: λ Int+ LVA p22 attP Reverse Terminator p22 attB (rev comp) λXis +AAV ECFP +AAV λHalf Bit BBa_I11060 : BBa_I11020 BBa_I11033 BBa_B0025 BBa_I11032 BBa_I11021 BBa_E0024 p22 Int+ LVA λattP Terminator λattB (rev comp) P22 Xis +AAV EYFP +AAV p22 Half Bit BBa_I11061 : BBa_I11030 BBa_I11023 BBa_B0013 BBa_I11022 BBa_I11031 BBa_E0034

21. Synthesis & Testing: Can Int + Xis control GFP expression? PLlacO PLtetO attP Int GFP_AAV attB* pBAD Xis pSC101 Kan • Lutz and Bujard, Nuc. Acids Res., 1997, Vol. 25, No. 6 1203-1210

22. * = variable region RBSI x2 TagI x3 RBSX x2 TagX x3 S/FP ‘Read-out’ x2 I-X Pairs x5 HUGE Increase in Complexity 360 New Test Constructs Trouble-shooting the Int/Xis Counter • No detectable GFP expression • attP sterically hinders expression? • Solution: Swap positions of attB & attP • Potential problems with plasmid copy numbers • Noise effects & cross recombination b/w plasmids • Solution: Integrate a single-copy into the genome via λ red recombination • Need more variants to better characterize the system Solution: Multiplex DNA Synthesis

23. Integrating Multiplex DNA Synthesis & Synthetic Biology Identify Desired Sequences Implement software to design oligos for multiplex DNA synthesis Parallel Construction of ALL new constructs via multiplex DNA synthesis Integrate Constructs into E. coli genome via λ red recombination High throughput Screening & Selection Experiments to isolate desired behavior

24. Acknowledgements Harvard University John Aach Patrik D'haeseleer Gary Gao Hui Gong Jinkuk Kim Xiaoxia Lin Jingdong Tian Sasha Wait Nathan Walsh George Church Boston University Will Blake Jim Flanigon Ellen O’Shaughnessy Margot Schomp Jim Collins MIT Peter Carr Chris Emig Joe Jacobson Farren Isaacs: farren@genetics.med.harvard.edu