& Implications for Applied Microbiology Janet Nguyen Andrei Anghel Nagham Chaban Synthetic Life & Implications for Applied Microbiology 1 1 1

What is Synthetic Biology? Definition: Synthetic biology is the engineering of biology; the synthesis of complex, biological based system, which display function that does not exist in nature. In essence, synthetic biology enables the design of biological system in rational and systemic way Drafted by the NEST High Level Expert Group New and Emerging Science and Technology (NEST) High-Level Expert Group 2 2 2

Overview of Presentation J. Craig Venter Institute: Synthesized a novel 1.1 mbp genome Transplanted a synthetic genome into host cells and completely replaced the host genome New cells were capable of self-replication and expressed only novel genes 4 4 4

Overview of Presentation Topics to be Covered: Genome Synthesis Intercellular Transplant Potential Uses of Technology 5 5 5

Timeline of Advancements 6 6

Experimental Organisms Organisms were specifically chosen for: Size of genome Stability of genome in host Speed of replication Lack of cell wall 7 7 7

Experimental Organisms Donor: Mycoplasma mycoides Subspecies: mycoides Strain: Large Colony GM12 Replicates every 80 min Recipient: Mycoplasma capricolum Subspecies: capricolum Strain: California Kid (CK) Replicates every 100 min 8 8 8

Experimental Organisms Mycoplasma genus 9 9 9

1. Genome Synthesis Janet Nguyen 10 10 10 10

Synthesis: Designing the genome M. mycoides JCVI-syn1.0 Biologically significant differences were corrected Synthetic and wild type polymorphic at 19 sites Watermark sequences Sequences encode unique identifiers Limits their translation into peptides Watermark sequences: Replace regions experimentally demonstrated to not interfere with cell viability Differences at 19 sites appeared harmless and were not corrected 11

Synthesis: Interesting watermarks A code to interpret the rest of the watermarks and website address. To live to err, to fall, to triumph, to recreate life out of life. See things not as they are, but as they might be. What I cannot build, cannot understand. 3 months to find single bp deletion error 2008 genome - simply signed the names of contributing scientists as watermarks 2010 - With first genome, were criticized for trying not to say something more profound than signing the works. Thus if the DNA sequence is GTTCGA, then GTT will be one letter and CGA will be another, and so on. 12

Synthesis: The Genome Mycoplasma mycoides JCVI-syn1.0 Watermark sequences: Replace regions experimentally demonstrated to not interfere with cell viability Differences at 19 sites appeared harmless and were not corrected 13

Synthesis Overview 1. 1 kb fragments 2. 10 kb fragments 4. Complete genome 14

Synthesis: Strategy Hierarchical strategy: 3 Stages 1 kb → 10 kb → 100 kb → genome (1000 kb) Start with 1 kb fragments (n=1078) with 80 bp overlaps to join to neighbours chemically synthesized by Blue Heron have restriction enzyme sites at termini each cassette was individually synthesized and sequence-verified by manufacturer To aid in building process, DNA cassettes and assembly intermediates were designed to contain restriction sites at their termini, and recombined in the presence of vector elements to allow for growth and selection in yeast Hierarchical strategy was designed to assemble the genome in three stages by transformation and homologous recombination in yeast from 1078 1-kb cassettes. 15

Synthesis: Stage 1 = 1 kb to 10 kb 1-kb fragments and a vector recombined in vivo in yeast Very active recombination system! Plasmid then transferred to E.coli Generally, at least one 10-kb assembled fragment could be obtained by screening 10 yeast clones. But rate of success varied from 10-100% Plasmid DNA isolated form E.coli clones: digested to screen for cells containing a vector with an assembled 10-kb insert 19/111 had errors.Alternate clones selected, sequence-verified before next stage 16

Synthesis: Stage 1 = 1 kb to 10 kb 1-kb fragments and a vector recombined in vivo in yeast Very active recombination system! Plasmid then transferred to E.coli Generally, at least one 10-kb assembled fragment could be obtained by screening 10 yeast clones. But rate of success varied from 10-100% Plasmid DNA isolated form E.coli clones: digested to screen for cells containing a vector with an assembled 10-kb insert 19/111 had errors.Alternate clones selected, sequence-verified before next stage 17

- all you need to add here is a origin of replication for yeast, and a yeast chromsomal centromere because it is eukaryotic. Turning it into a shuttle vector and it can propagate in either E.coli or yeast.

Synthesis: Stage 1 = 1 kb to 10 kb Recombinant plasmid isolated from E.coli clones Plasmids digested to find cells with assembled 10 kb insert All first-stage assemblies sequenced 19/111 had errors End of Stage 1: results in 10-kb fragments (n=109) Generally, at least one 10-kb assembled fragment could be obtained by screening 10 yeast clones. But rate of success varied from 10-100% Plasmid DNA isolated form E.coli clones: digested to screen for cells containing a vector with an assembled 10-kb insert 19/111 had errors.Alternate clones selected, sequence-verified before next stage 19

Synthesis Overview 1. 1 kb fragments 2. 10 kb fragments 4. Complete genome 20

Synthesis: Stage 2 = 10 kb to 100 kb 10 kb fragments and cloning vectors transformed into yeast 100 kb assemblies not stably maintained in E.coli Recombined plasmid extracted from yeast Multiplex PCR presence of a PCR product would suggest an assembled 100 kb PCR products run on agarose gel End of Stage 2: Results in 100 kb fragments (n=11) Explain PCR: Since every 10-kb assembly intermediate was represented by a primar pair, the presence of all amplicons would suggest an assembled 100-kb intermediate. (generally, 25% or more of the clones screened contained all of the amplicons expected for a complete assembly). Designed primer to represent a 10-kb fragment so that the presence of the PCR product would suggest an assembled 100-kb piece. After PCR: One of these clones selected for further screening. Circular plasmid DNA was extracted and sized on agarose gel alongside supercoiled marker. Successful second-stage assemblies with vector sequence are ~105 kb When all amplicons were produced following multiplex PCR, second-stage assembly intermediate of the correct size was usually produced In some cases, small deletions occurred. In others, multiple 10-kb fragments were assembled, which produced a larger second-stage assembly intermediate. These differences were easily detected on an agarose gel before complete genome assembly. 21

Synthesis Overview 1. 1 kb fragments 2. 10 kb fragments 4. Complete genome 22

Synthesis: Complete genome assembly Isolated small quantities of each 100 kb fragment Purification: exonuclease then anion-exchange column Small fraction of total plasmid DNA (1/100) was digested Then analyzed by gel electrophoresis Result: 1ug of each assembly per 400ml of yeast culture Not all yeast chromosomal DNA removed After first point: (Circular plasmids the size of the second-stage assemblies could be isolated from yeast spheroplasts after an alkaline-lysis procedure). To further purify the 11 assembly intermediates, they were treated with exonuclease and passed through an anion-exchange column. .A small fraction of the total plasmid DNA, 1/100, was digested with NotI and analyzed by field-inversion gel electrophoresis. This method produced ~1ug of each assembly per 400 ml of yeast culture, ~10^11 cells. Method above does not completely remove all linear yeast chromosomal DNA, which could substantially decrease yeast transformation and assembly efficiency 23

Synthesis: Complete genome assembly To further enrich for the 100 kb fragments: Sample of each fragment mixed with molten agarose As agarose solidifies, fibers thread and “trap” circular plasmids Trapped plasmids digested, releases inserts gel electrophoresis transformed into yeast, no vector sequence required Complete genome assembled in vivo in yeast, and grown as yeast artificial chromosome Untrapped linear DNA can be separated out of the agarose plug by electrophoresis, thus enriching for the trapped circular molecules. In this third/final stage of assembly, an additional vector sequence was not required b/c the yeast cloning elements were already present in assembly 811-900. 24

Synthesis complete! Transplantation of genome Verification of genome Next steps: Transplantation of genome Verification of genome

2. Intercellular Transplantation Andrei Anghel 26 26 26 26

Transplant Overview Dr. Carole Lartigue 27 27

Transplantation: Hurdles Genetic modifications made to DNA template in order to allow cloning entire chromosome as a plasmid in yeast Inactivation of recipient cell restriction enzyme Methylation of the synthetic genome 28 28 28

Transplantation: Hurdles Genetic modifications made to DNA template in order to allow cloning entire chromosome as a plasmid in yeast Inactivation of recipient cell restriction enzyme Methylation of the synthetic genome 29 29 29

Transplantation: Hurdles Genetic modifications made to DNA template in order to allow cloning entire chromosome as a plasmid in yeast Inactivation of recipient cell restriction enzyme Methylation of the synthetic genome 30 30 30

Transplantation: Hurdles Genetic modifications made to DNA template in order to allow cloning entire chromosome as a plasmid in yeast Inactivation of recipient cell restriction enzyme Methylation of the synthetic genome 31 31 31

Transplantation: Procedure Starved M. capricolum cells were mixed with isolated, synthetic DNA Incubated for 3 hours at 37°C to allow recovery, then plated until large blue colonies formed Blue colonies were then used to inoculate selective broth tubes 32 32 32

The Complete Synthetic Cell

The Complete Synthetic Cell

Transplantation: Verification and Efficiency Ensuring no false-positive results was crucial M. mycoides JCVI-syn1.0 was transformed with a vector containing a selectable tetracycline-resistance marker and a b-galactosidase gene for screening PCR experiments and Southern blot analysis of isolated putative transplanted cells Multiple specific antibody reactions were carried out to test for species specific proteins 35 35 35

Transplantation: Verification 36 36 36

Transplantation: Verification and Efficiency Only 1 out of 48 yeast colonies contained a full genome Only 1 in 150,000 successful transplants in the most efficient experiments Transplant yield was optimal with 107 - 5×107 cells used Yields began to plateau at high donor DNA concentrations 37 37 37

3. Potential Uses of the Technology Nagham Chaban 38 38 38 38

Uses of the Technology DNA is the software of life How could synthetic biology and DNA transfer affect our lives? Creating synthetic bacteria and transferring man-made DNA allowed the new bacteria to live and replicate That was proof of principle that life can be created from a computer 39 39 39

Uses of the Technology Designing synthetic bacteria ensures that synthetic DNA can be used for valuable things in our lives The key is to understand how to change this software in order to create synthetic life Can lead to powerful technology and many applications and products: biofuel, medicines, food, etc. 40 40 40

Applications: Medicine MALARIA Kills many people Numerous malaria pathogens are resistant to the first generation drug Artemisinin is a second generation drug that can treat malaria But there is always a problem! 41 41 41

Applications: Medicine Artemisinin is available in low quantity in nature Synthetic biology can be the solution by building up a new biosynthetic pathway for this molecule in microorganisms (i.e. yeast or E.coli) 42 42 42

Applications: Medicine THERAPEUTIC BACTERIA Strange idea, we think of bacteria to be associated with disease, not therapy TUMOR-KILLING BACTERIA Creating a safe synthetic bacteria to be injected into the bloodstream Travel to tumor, insert itself into cancer cell, produce tumour-killing toxin 43 43 43

Applications: Food Products ACTIVIA People are infecting themselves with bacteria Can improve digestion People like this! 44 44 44

Applications: Energy Production BIOFUELS Important issue worldwide Plants  biofuels Plant biomass simple sugars Fermented sugar  energy 45 45 45

Applications: Risks Natural genome pool contamination Synthetic products released in the environment should have a specific life span Creation of deadly pathogens: bio-terrorism Negative environmental impact Global monitoring and tracking of synthetic products are necessary 46 46 46

Overview ~1 million bp synthetic genome Synthetic genome was transplanted into a cell of a different subspecies – booted up! Vast implications/uses for applied microbiology Synthetic biology can reshape our lives and transfer our society Important concerns regarding religion (playing with god) should be discussed and addressed 47 47 47

Thank you for staying awake Questions & Ethics Discussion Thank you for staying awake 48 48 48 48

Discussion Points What if a synthetic RNA can be designed to catalyze its own reproduction within an artificial membrane? No guarantee that a synthetic genome that works for one organism (E. coli) will work in another (B. subtilis) Cost/expenses Religious/ethical issues 49 49 49

References Gibson, D. G., Glass, J. I., Lartigue, C., Noskov, V. N., Chuang, R., Algire, M. A., et al. (2010). Creation of a bacterial cell controlled by a chemically synthesized genome. Science, 329(5987), 52-56. Lartigue, C., Glass, J. I., Alperovich, N., Pieper, R., Parmar, P. P., Hutchison III, C. A., et al. (2007). Genome transplantation in bacteria: Changing one species to another. Science, 317(5838), 632-638. Laitigue, C., Vashee, S., Algire, M. A., Chuang, R. -., Benders, G. A., Ma, L., et al. (2009). Creating bacterial strains from genomes that have been cloned and engineered in yeast. Science, 325(5948), 1693-1696. 50 50 50 50