From oligos to organisms

Transcription

1 From oligos to organisms A synthetic biology approach to systems biology Mikkel Algire for the The Synthetic Biology and Bioenergy Group J. Craig Venter Institute

2 Biological life is complex interactions Genomic DNA RNA Coding and noncoding (mrna, rrna, trna, srna, etc.) Living cell proteins small molecules lipids

3 Biological life is complex interactions Genomic DNA RNA Coding and noncoding (mrna, rrna, trna, srna, etc.) Living cell proteins small molecules lipids How can synthetic biology contribute to furthering the understanding of this complex system?

4 Synthetic biology tools DNA assembly s of base pairs (genes, plasmids) to Entire genomes (580 kb) In vitro E. coli Yeast Genome transplantation Nucleotide level control of entire genome - Minimial genomes - Metabolic manipulation - and much more

5 Synthetic biology tools DNA assembly Chew-back anneal (CBA) DNA assembly In vitro homologous recombination Starting oligos 300 bp dsdna 5 overlapping 300 bp DNAs 1500 bp dsdna *Fast and efficient *Can produce DNA ranging from genes to genomes *Total control of sequence Larger molecules

6 Synthetic biology tools DNA assembly Chew-back anneal (CBA) DNA assembly In vitro homologous recombination DNA fragments can be produced by PCR Starting oligos 300 bp dsdna 5 overlapping 300 bp DNAs 1500 bp dsdna *Fast and efficient *Can produce DNA ranging from genes to genomes *Total control of sequence Larger molecules

7 The nuts and bolts of Chew-Back Anneal (CBA) -Fast - One step isothermal method, 50 C, ~30 mins 5 3 Homology between DNAs (25-40 bp) DNA 1 DNA Chew back with T5 exonuclease (T5 exo is inactivated in 15 min at 50 C) Anneal, fill in and ligate (Phusion polymerase, Taq ligase) Usually performed in the presence of a vector backbone to create a circular DNA

8 One-step isothermal in vitro recombination *Enzymes and reagents are commercially available *Assembly master mixture can be stored frozen E. coli *40 bp overlaps *Clone > 300kb in E. coli

9 Nature Methods Enzymatic assembly of DNA molecules up to several hundred kilobases. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO. Nat Methods May;6(5): See Supplementary Methods and Nature Protocols online

10 What can you do with CBA? Synthesize any desired sequence using oligos, PCR amplicons, natural DNA fragments or a combination of these DNAs. For example we have *Created a yeast-bacteria-mycoplasma Trishuttle vector *Synthesized methyltransferase genes and assembled into expression vectors. *Changed promoter sequences and selection markers *Can be used to perform efficient site-directed mutagenesis *Create any plasmid you want from PCR products *Synthesized a Mycoplasma genitalium genome

11 What can you do with CBA? Synthesize any desired sequence using oligos, PCR amplicons, natural DNA fragments or a combination of these DNAs. For example we have *Created a yeast-bacteria-mycoplasma Trishuttle vector *Synthesized methyltransferase genes and assembled into expression vectors. *Changed promoter sequences and selection markers *Can be used to perform efficient site-directed mutagenesis *Create any plasmid you want from PCR products *Synthesized a Mycoplasma genitalium genome

12 Q: Why synthesize a M. genitalium genome?

13 Q: Why synthesize a M. genitalium genome? A: To produce a minimal cell.

14 Q: Why synthesize a M. genitalium genome? A: To produce a minimal cell. Q: What is a minimal cell?

15 Q: Why synthesize a M. genitalium genome? A: To produce a minimal cell. Q: What is a minimal cell? A: A cell is minimal under ideal laboratory conditions if (1) it can be grown in pure (axenic) culture (2) each and every one of its genes is essential.

16 Q: Why the interest in such a cell? *Biology has always made great advances using simple systems. A minimal cell would be an ideal platform for exploring the nature of cellular life. *If the function of every gene is determined, then it may be possible E. to coli achieve a complete understanding of what Minimal it cell takes to be alive. More complex Less complex *It may be possible to model the minimal cell s behavior on a computer and determine the effects of environmental variations or Genomic Genomic the effects DNA of added metabolic pathways, etc.. DNA *A minimal cell can be used as a launching pad for making more complex and RNA useful organisms RNA Coding and noncoding Coding and noncoding *The capacity to synthesize cells could allow humans to design (mrna, rrna, trna, srna, etc.) (mrna, rrna, trna, srna, etc.) organisms with extraordinary properties that could solve energy problems, sequester CO 2, or produce pharmaceuticals and industrial compounds proteins proteins small molecules lipids small molecules lipids

17 Q: Why the interest in such a cell? *Biology has always made great advances using simple systems. A minimal cell would be an ideal platform for exploring the nature of cellular life. *If the function of every gene is determined, then it may be possible to achieve a complete understanding of what it takes to be alive. *It may be possible to model the minimal cell s behavior on a computer and determine the effects of environmental variations or the effects of added metabolic pathways, etc.. *A minimal cell can be used as a launching pad for making more complex and useful organisms *The capacity to synthesize cells could allow humans to design organisms with extraordinary properties that could solve energy problems, sequester CO 2, or produce pharmaceuticals and industrial compounds

18 Q: Why the interest in such a cell? *Biology has always made great advances using simple systems. A minimal cell would be an ideal platform for exploring the nature of cellular life. *If the function of every gene is determined, then it may be possible to achieve a complete understanding of what it takes to be alive. *It may be possible to model the minimal cell s behavior on a computer and determine the effects of environmental variations or the effects of added metabolic pathways, etc.. *A minimal cell can be used as a launching pad for making more complex and useful organisms *The capacity to synthesize cells could allow humans to design organisms with extraordinary properties that could solve energy problems, sequester CO 2, or produce pharmaceuticals and industrial compounds

19 Q: Why the interest in such a cell? *Biology has always made great advances using simple systems. A minimal cell would be an ideal platform for exploring the nature of cellular life. *If the function of every gene is determined, then it may be possible to achieve a complete understanding of what it takes to be alive. *It may be possible to model the minimal cell s behavior on a computer and determine the effects of environmental variations or the effects of added metabolic pathways, etc.. *A minimal cell can be used as a launching pad for making more complex and useful organisms. *The capacity to synthesize cells could allow humans to design organisms with extraordinary properties that could solve energy problems, sequester CO 2, or produce pharmaceuticals and industrial compounds.

20 Q: Why chose M. genitalium? *Has the smallest genome (580 kb) of any known bacterium capable of independent life. *485 protein coding genes and 43 RNA genes *115 of its 485 protein coding genes can be disrupted Unknown how many of these genes are simultaneously dispensable

21 Approach used to synthesize a M. genitalium cell Assemble overlapping synthetic oligonucleotides (~60 mers) Recipient cell Synthetic cell Cassettes (5-7 kb) Assemble cassettes by homologous recombination Genome transplant into recipient cell Completely assembled synthetic genome

22 Approach used to synthesize a minimal cell Assemble overlapping synthetic oligonucleotides (~60 mers) Recipient cell Cassettes (5-7 kb) Combinatorial assembly of cassettes by homologous recombination Pool of reduced genomes Minimized synthetic cells

23 Approach used to synthesize genome Assemble overlapping synthetic oligonucleotides (~60 mers) Cassettes (5-7 kb) Assemble cassettes by homologous recombination Modifications to the genome: 1. 5 Watermark sequences added to differentiate the synthetic genome from the native genome. Completely assembled synthetic genome 2. Disrupted a pathogenicity gene and replaced it with an antibiotic resistance marker to select for cells containing the synthetic genome.

24 Assembling a synthetic M. genitalium genome Synthetic genome sequence divided into 101 overlapping cassettes. Each cassette = 5 to 7kb M. genitalium chromosome ~580kb

25 Five-stage assembly strategy 6kb 24kb 72kb 144kb 290kb 580kb etc. 1/25 1/8 1/4 1/2 Whole

26 Assembling with 40bp overlaps makes cloning DNA fragments easy 40bp sequence found at 5 end of cassette #78 20bp BAC sequence Not I Restriction Sites BAC (8.2kb) Cm R 20bp BAC sequence 40bp sequence found at 3 end of cassette #81 B B Simultaneous assembly with a PCR-amplified BAC 78 B Directly Transform E. coli Purpose: To make more of the assembled synthetic cassettes for subsequent rounds of assembly

27 Series Cloning the A, B, and C-series assemblies No. fragments assembled Approximate assembled insert size Typical # colonies Percent colonies with correct insert size 1/ kb / kb / kb BAC 1/25 1/8 1/4 C25-49 B50-61 A78-81 Released BAC 146 kb 97 kb 48.5 kb 23 kb 9.4 kb 6.6 kb Not I digestion to release inserts

28 E. coli could only take us so far. 6kb X X 24kb 72kb 144kb 290kb 580kb etc. 1/25 1/8 1/4 1/2 Whole

29 The entire M. genitalium genome could be assembled in vitro but E. coli clones did not have full-length molecules. Λ ladder 582 kb kb kb Whole chromosome ¾ molecules ½ molecules No E. coli clones kb E. coli clones In vitro DNA assembly

30 How are we going to clone a completely assembled M. genitalium genome? Solution: Clone in Saccharomyces cerevisiae Size limit for cloning in yeast is 7X that of E. coli (~ 2Mb) Can we use yeast s powerful homologous recombination system to assemble our overlapping pieces in vivo?

31 E. coli 1 E. coli 2 E. coli 3 E. coli 4 Isolate ¼ molecules from E. coli Digest with Not I to release inserts Combine with vector containing yeast selectable marker/centromere 4 X 1 3a BsmBI 3b X 2 Transform mixture into yeast Cloning vector yeast

32 The synthetic M. genitalium genome has been cloned! The sequence exactly matched our designed genome! M. gen genome Not I 582 kb N N M 48.5 kb Science Feb 29;319(5867):

33 A synthetic M. genitalium genome has been assembled in four stages 6kb 24kb 72kb 144kb 580kb yeast Yeast Vector 50 77A 50 77B 1/25 1/8 1/4 Whole E. coli yeast

34 How about two stages? 6kb 24kb 580kb A86-89 has internal vector 1/25 Whole Can a single yeast cell take up 25 pieces?

35 Can 25 pieces be assembled in yeast in one shot?

36 Can 25 pieces be assembled in yeast in one shot? YES! Yeast cloning and recombination is a powerful tool for synthetic biology Proc Natl Acad Sci U S A Dec 23;105(51):

37 The synthetic M. genitalium genome in yeast Now what? M. gen genome

38 Synthetic biology tools DNA assembly s of base pairs (genes, plasmids) to Entire genomes (580 kb, 1.1 Mb) In vitro E. coli Yeast Genome transplantation Nucleotide level control of entire genome - Minimial genomes - Metabolic manipulation - and much more

39 Genome transplantation First we showed we could isolate native M. mycoides genomic DNA and transplant it into M. capricolum. naked genomic DNA M. mycoides (Tet R ) Cells arising from genome transplantation have the genotype and the phenotype of the M. mycoides donor. M. capricolum (Recipient cell) M. mycoides (Transplant cell) (Tet R ) Science Aug 3;317(5838):632-8.

40 Genome transplantation In order to create a synthetic cell we need to be able to transplant a bacterial genome isolated from yeast. naked genomic DNA M. mycoides (Tet R ) M. capricolum (Recipient cell) M. mycoides (Transplant cell) (Tet R ) Yeast cell with cloned bacterial genome

41 Genome transplantation Issues: Only genome we have been able to transplant is M. mycoides. M. genitalium synthetic genome is the one cloned in yeast. naked genomic DNA M. mycoides M. capricolum (Recipient cell) M. mycoides (Transplant cell) M. genitalium genome Yeast cell with cloned bacterial genome

42 Genome transplantation Clone M. mycoides genome in yeast and use as donor genome. Insert yeast selectable marker and centromere naked genomic DNA M. mycoides M. mycoides genome Yeast cell Yeast cell with genome

43 Bacterial genome engineering in yeast Prior to transplantation of the M. mycoides genome from yeast we used the powerful set of yeast tools to engineer the M. mycoides genome in a way that is impossible with current mycoplasma tools. Seamless deletion of a nonessential Type III restriction endonuclease gene. The resulting M. mycoides genome has never existed in a living bacterial cell.

44 Genome transplantation Can we do it? M. mycoides genome Yeast cell with engineered M. mycoides genome naked M. mycoides genomic DNA M. capricolum (Recipient cell) M. mycoides (Transplant cell)

45 Genome transplantation Genome and biochemical analysis revealed the presence of restriction-modification systems in both the donor and recipient. M. mycoides GATC CCTTC TGAG GANTC GCATC CCATC Yeast cell with M. mycoides genome CH 3 CH 3 protected degraded CH 3 CH 3 modified M. mycoides genomic DNA M. capricolum (Recipient cell) CCATC unmodified M. mycoides genomic DNA

46 Overcoming restriction barrier Donor-centric method Cytoplasmic extract of native M. mycoides cells. GATC CCTTC TGAG GANTC GCATC CCATC Unknowns? M. mycoides Synthesize and express methyltransferases in E. coli. Isolate genome from yeast unprotected GATC CCTTC TGAG GANTC GCATC CCATC Purified methyltransferases CH 3 CH 3 CH 3 CH 3 Protected genome

47 Overcoming restriction barrier Recipient-centric method Inactivate active restriction endonuclease in recipient. Targeted single-crossover into endonuclease gene X M. capricolum (Recipient cell) M. capricolum RE

48 Overcoming restriction barrier Recipient-centric method Inactivate active restriction endonuclease in recipient. Targeted single-crossover into endonuclease gene X M. capricolum (Recipient cell) M. capricolum RE We attempted all three methods: extracts Purified methyltransferases Endonuclease inactivation in recipient

49 Genome transplantation Yeast cell with engineered M. mycoides genome CH 3 CH 3 M. capricolum In vitro methylation CH 3 CH 3 Transplant into both recipient cells M. mycoides genomic DNA M. mycoides (Transplant cell) X M. capricolum RE (Recipient cells)

50 M. Mycoides genomic DNA isolated from yeast can be transplanted into M. capricolum!!! Methylation treatment Number of transplants (colonies/plug) Wild-type M. capricolum recipient M. capricolum RE recipient untreated 0 37 ± 3 M. mycoides extracts M. mycoides purified methylases M. capricolum extracts 22 ± 8 15 ± 8 13 ± ± 17 9 ± 4 32 ± 13 Mock-methylated 0 34 ± 17 a Average of at least 3 experiments. The error reported is the mean deviation. Only needed to protect DNA from recipients restriction systems. Science Sep 25;325(5948):

51 Producing bacteria from genomes engineered in yeast Overlapping DNA fragments (natural or synthetic) and a yeast vector

52 Producing bacteria from genomes engineered in yeast Overlapping DNA fragments (natural or synthetic) and a yeast vector Applications: 1. Combinatorial genome reduction to construct a minimal cell 2. Intelligently redesign intractable organisms and microbes for which there are poor genetic tools

53 Synthetic biology take-home lessons *With CBA DNA assembly and in vivo yeast recombination you can easily synthesize any sequence you can specify from genes to genomes. *DNA and genome construction is NOT going to be the rate limiting step in producing designer cells. *It will soon be possible to synthesize any sequence and install it in a cell where it can be expressed. *Work on computational tools for genome and pathway design is urgently needed.

54 The Synthetic Biology and Bioenergy Group at JCVI - October 2007 JCVI La Jolla, CA JCVI Rockville, MD

55 Life by Intelligent Design Clyde Hutchison Ham Smith Craig Venter Clyde Hutchison

56 It takes a village to create a cell Algire, Mikkel Alperovich, Nina Assad-Garcia, Nacyra Baden-Tillson, Holly Benders, Gwyn Chuang, Ray-Yuan Dai, Jianli Denisova, Evgeniya Galande, Amit Gibson, Daniel Glass, John Hutchison, Clyde Iyer, Prabha Jiga, Adriana Krishnakumar, Radha Lartigue, Carole Ma, Li Merryman, Chuck Montague, Michael Moodie, Monzia Moy, Jan Noskov, Vladimir Pfannkoch, Cindi Phang, Quan Qi, Zhi-Qing Ramon, Adi Saran, Dayal Smith, Ham Tagwerker, Christian Thomas, David Tran, Catherine Vashee, Sanjay Venter, J. Craig Young, Lei Zaveri, Jayshree Johnson, Justin Brownley, Anushka Parmar, Prashanth Pieper, Rembert Stockwell, Tim Sutton, Granger Viswanathan, Lakshmi Yooseph, Shibu Funding from Synthetic Genomics Inc. JCVI DOE GTL program