Synthetic  Biology  – Trends  and  UpdatesEC  WORKSHOP  ON  SYNTHETIC  BIOLOGY  -­‐

FROM  SCIENCE  TO  POLICY  AND  SOCIETAL  CHALLENGESLuxembourg  9th Dec  2015  

Professor  Paul  Freemont    @paulfreemontCo-­‐director  and  Co-­‐founder  EPSRC  Centre  for  Synthetic  Biology  and  InnovationCo-­‐director  and  Co-­‐founder  of  UK  National  Innovation  and  Knowledge  Centre  for  Synthetic  Biology  Imperial  College  London,  UK

WHAT  IF  ?

E.  chromi,  2009  University  of  Cambridge  iGEM team  

WHAT  IF  ?

Plasticity  2013  Imperial  College   iGEM team  http://2013.igem.org/Team:Imperial_College  

WHAT  IF  ?

Aqualose 2014   Imperial  College   iGEM team    http://2014.igem.org/Team:Imperial

Gluconacetobacter xylinus

Gluconacetobacter xylinus +  yeast

Suzanne  Lee

Imperial  College   IGEM    2014  Aqualose

Genetic  engineering  of  the  cellulose-­‐producing  bacterium  Komagataeibacter rhaeticus for  production  of  novel  biomaterials.Florea et  al      2015  in  press

WHAT  IF  ?  

viruses

parasites

bacteria

Infector  Detector    2007   Imperial  College   iGEM team    http://2007.igem.org/Imperial/Infector_Detector/Introduction

coal

petroleum

natural gas

Carbon feedstocks Building blocks Value - added chemicals

ethylene / propylene / butadiene

benzene / toluene / xylene

WHAT  IF  ?

Building blocks Value - added chemicals

ethylene / propylene / butadiene

benzene / toluene / xylene cellulosic crops

Biomass feedstocks

CO2

lignocellulosicwaste

WHAT  IF  ?

S.  cerevisiae secreting   farnesene/biodiesel

~112K  bases  added~41K  bases  removed~450  single  nucleotide  changes

~1.25%  of  the  genome!

DNA  as  a  programmable  material  

WHAT  IF  ?  Eau  de   Yeast

GenSpace’s

DIY-­‐Bio,  Biohackers  and    the  Growth  of  Community  Labs

(adapted  from  Science  333:  6047  (2011);  http://www.wisegeek.com/what-­‐are-­‐cold-­‐forceps.htm);  http://www.webmd.com/colorectal-­‐cancer/ss/slideshow-­‐colorectal-­‐cancer-­‐overview)

Engineered  bacteria  to  detect  and  kill  cancer  cells

Engineered  E.  coliColorectal  cancer

Healthy  colon

WHAT  IF  ?  

(adapted  from  Science  333:  6047  (2011);  http://www.sciencephoto.com/)

bacteriophageAttacking  bacterium Bacteria  cell  lyses  and  dies

Engineered  phage  and  bacteria  to  target  pathogens

Engineered  E.  coli

WHAT  IF  ?  

Porter  et  al.  N.  Eng.  J.  Med.,  365  (2011),  pp.  725–733

T  cell  engineering

Engineered  T-­‐cells  to  target  cancerWHAT  IF  ?  

Warren  et  al.  Cell  Stem  Cell  7,  618  (2010)

Cell  therapy  and  regenerative  medicine

Systematic  cellular  reprogramming  

Induced  pluripotent  stem  cells

WHAT  IF  ?  

October  2015

WHAT  IF  ?

https://www.youtube.com/watch?v=ylSZN-­‐1hhoY

“Synthetic  biology  aims  to  design  and  engineer  biologically  based  parts,  novel  devices  and  systems  as  well  as  redesigning  existing,  natural  biological  systems”

So  why  is  synthetic  biology  causing  such  a  big  fuss?  

“SynBio is  the  application  of  science,  technology  and  engineering  to  facilitate  and  accelerate  the  design,  

manufacture  and/or  modification  of  genetic  materials  in  living  organisms.”

SCHER,  SCENIHR,  SCCSoperational  definition  for  Synthetic  Biology

Synthetic  Biology  is  a  rapidly  growing  field

Citations    of  papers  containing  key  works  synthetic  biology    70256    -­‐ 47,000  papers  since  2001  

A  growing  community  of  student  researchers  – growth  of  iGEMInternational  Genetically  Engineered  Machine  Competition

280  teams  are  registered  for  2015259  teams  at  the  Jamboree~15,000  iGEM alumni  

Synthetic  Biology  has  a  powerful  vision  for  merging  engineering  design  practice  into  the  construction  of  

biology  systems  and  cells  at  the  genetic  level

In  engineering  systems,  robustness  and  stability    are  achieved  by  

(1) System  control(2) Redundancy(3) Modular  design(4)  Structural  stability

Basics  of  an  engineering   design  frameworkBasics  of  an  engineering   design  framework

(1) System  control  (feed-­‐back/   feed-­‐forward  biological  control  networks  )(2) Redundancy (gene  duplication/  multiple  regulatory  pathways  )(3) Modular  design  (evolutionary  robust  /  multi-­‐functional  /    compartmental)(4)  Structural  stability   (homeostasis)

Hypothesis  – Are  these  also  intrinsic  features  of  complex  natural  living  systems?

A  systematic  engineering  framework  for  biological  systems  aims  to  test  the  hypothesis

An  engineering  design  framework  for  Synthetic  Biology  

An  engineering  design  framework  for  Synthetic  Biology  

Can  we  use  Biology  to  Build  new  Biology?Can  we  learn  about  biology  through  

design  and  construction?

• Biological  systems  are  modular  • Biological   function  is  primarily  encoded  in  DNA• Large  knowledge  base  of  genome  sequences• Large  diversity  of  biological  parts  (genes/regulatory)• Increased  understanding  of  molecular  / cell  biology• New  technologies  to  synthesize  and  assemble  DNA

However……

Standardising biology  poses  challenges

• Biology  is  not  fully  ‘plug  and  play’– Context  dependency– Evolution,  adaptation  and  natural  selection– Non-­‐predictive  stochastic  behaviour– Self  assembly  and  emergent  properties– Non-­‐linear  dynamical  processes  –Multi-­‐scale  interactions

• Living  cells  have  constrained  volumes  and  high  concentrations  of  biochemical  components

x

One  approach  to  overcome  biological  complexity  in  

engineering  biology  is  the  use  of  Systematic  Design  

What  is  Systematic  Design  ?  Systematic  design  is  founded  on  the  following  

engineering  principles

• Modularisation – interchangeable modules• Standardisation – standard parts  and  processes• Abstraction  – reducing  complexity

Systematic    design  aims  to  achieveRobustness  and  Reproducibility

Key  requirement  is  interoperability  

A  systematic  design   framework  using  genetic  parts  that  encode  biological   function

DNA

Parts

Devices

Systems

ATCGGTCAAGTGCCT

lacI

SLacI

PlaclacI

SLacI

Plac

hrpR

hrpS

R

S

R

S PhrpL

hrpR

hrpS

R

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R

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Typical  gene  transcription  module

Ribosome  binding  site

Protein  coding  sequence

Terminator

Transcription  factorTF

TF protein

promoter gene

Abstraction  hierarchy

Modularity

ResponsibleResearch  &  Innovation

A  systematic  DESIGN  CYCLE  for  Synthetic  BiologyA  systematic  DESIGN  CYCLE  for  Synthetic  Biology

Design

Build

Specification

Parts

DNA  Assembly

Test

Devices  &  Systems

Learn

Can  we  build  new  biological  systems  with  standardised DNA  Parts?

To  enable  forward  engineering   the  synthetic  biology  field  needs   to  develop  standards

The  first  standard  thread Sir  Joseph  Whitworth  1841

How  do  we  standardise the  construction    of  living  matter?

E.  coli Bacillus  megateriumB.  subtilis

Standards  development  in  synthetic  biology  

• Standard  interchangeable  biological  parts

• Physical  standards  (DNA)-­‐ Assembly  standards  (may  not   be  needed   with   increasing   DNA  synthesis)

• Functional  standards-­‐ Standard  culture  conditions  (media/temp/volume)-­‐ Standard  measurements  (e.g.  Flow  cytometry)-­‐ Standard  strains  of  cell  hosts  or  chassis

• Digital  Information  standards-­‐SBOL-­‐SBML-­‐DICOM-­‐SB  

Synthetic  Biology  Foundational  Technology  

Different  applications

Part  /  DeviceCharact.

Bio-­‐CAD  Design  tools

Chassis/  Host  cellCharact.

Genomeediting  /screens

DNA  Synthesis  And    Assembly  

Current  Synthetic  Biology  research   trends

• Engineering  of  biological  systems– Refactoring  and  Redesigning– Genome  editing– Genome  construction– Automation,  standards  and  tools– Deskilling  and  open  source

• Creating  alternative  biological  systems– exobiology/XNA– Artificial  cell  and  Cell  free  systems

Synthetic  Biology  Foundational  Technology  

Therapeutics&  Novel  Drug  Delivery  systems

FineSpeciality  Chemicals

Commoditychemicals

Biomaterials

Foundational  tools

Bio-­manufacturingprocesses

Agri-­Science

Synthetic  Biology  application  trends

DESIGN

Available  Bio-­‐Design  Tools  Pathway  and  circuit  designMATLAB:  Simbiology http://www.mathworks.co.uk/products/simbiology/OptCom http://maranas.che.psu.edu/software.htmGenetic  Engineering  of  Cells  (GEC)  http://research.microsoft.com/en-­‐us/projects/gec/Cell  designer    http://www.celldesigner.org/ProMoT http://www.mpi-­‐magdeburg.mpg.de/projects/promot/GenoCAD  http://www.genocad.org/Operon calculator  https://salis.psu.edu/software/OperonCalculator_EvaluateMode

Biopart designRosetta.  http://maranas.che.psu.edu/software.htmCadnano.  http://cadnano.org/NUPAC http://www.nupack.org/RNA  Designer  http://www.rnasoft.ca/cgi-­‐bin/RNAsoft/RNAdesigner/rnadesign.plmfold/UNAfold http://mfold.rna.albany.edu/RBS  Calculator  https://salis.psu.edu/softwareRBS  Designer  http://rbs.kaist.ac.krUTR  designer   http://sbi.postech.ac.kr/utr_designer

MiscellaneousR2oDNA  Designer  http://r2odna.com/SBOL http://www.sbolstandard.org/SBOLv http://www.sbolstandard.org/visual

Part  registries  worldwide

BUILD

Parts

Costs  of  DNA  synthesis  is  driving  the  field

Cost  per  base  – sequencing  ~0.000001  $– synthesis  ~0.10  – 0.28  $Rob  Carlson   http://www.biodesic.com/

Reading  DNA

Writing   genesWriting   short  

DNA

?

DNA  assembly  Standards    Tom  Ellis,  Geoff  Baldwin  

MODAL  – Modular  Overlap  Directed  Assembly  with  Linkers  

(A.  Casini et  al  NAR  2014a   and  2014b) P S

P S

P S1 2

P S2 3

P S3 4

P S4 1

P S

1

2 P S3

P

BASIC  -­‐ Biopart Assembly  Standard  for  Idempotent  CloningM.  Storch et  al  ACS  Synbio 4  :791  (2015)

part AP S

part BP S

part CP S

parts  ligation

part B

part C part A

Long-­‐overhang  assembly  No PCR!Robust  reactions  easy  to  automate

Interoperability  

Constructing  Synthetic  Yeast:  Sc2.0

www.syntheticyeast.orgwww.syntheticyeastresource.com

Jef Boeke (NY  Medical  School)

Design,  Synthesise  &  Assemble  a  modified  version  of  the  S.  cerevisiae genome  12  million  bp and  16  chromosomes

NYUUSA

BGICHINA

Johns   HopkinsUSA

JGIUSA

TianjinChina

EdinburghUK

ImperialUK

TsinghuaChina

MacQuarieAustralia

Aus  Wine  Res  InstAustralia

NUSSingapore

A  global  synthetic  biology  project

Sc2.0:  16  chromosomes,  12  million  bp

MacQuarie U

2014  – Completed  Syn Chromosome  III

Research Articles

/ http://www.sciencemag.org/content/early/recent / 27 March 2014 / Page 1 / 10.1126/science.1249252

Saccharomyces cerevisiae has a ge-nome size of ~12 megabases (MBs) distributed among 16 chromosomes. The entire genome encodes ~6000 genes of which ~5000 are individually nonessential (1). Which of these non-essential genes are simultaneously dis-pensable? While a number of studies have successfully mapped pairwise “synthetic lethal” interactions between gene knockouts, those methods do not scale well to 3 or more gene combina-tions because the number of combina-tions rises exponentially. Our approach to address this question is to produce a synthetic yeast genome with all nones-sential genes flanked by loxPsym sites to enable inducible evolution and ge-nome reduction (a process referred to as SCRaMbLEing) in vivo (2, 3). The availability of a fully synthetic S. cere-visiae genome will allow direct testing of evolutionary questions such as “what is the maximum number of nonessential genes that can be deleted without a catastrophic loss of fitness?” and “what is the catalog of viable 3-gene, 4-gene, … n-gene deletions that survive under a given growth condition?” that are not otherwise easily approachable in a sys-tematic unbiased fashion. Engineering and synthesis of viral and bacterial genomes have been reported in the literature (4–11). An international group of scientists, has embarked on constructing a designer eukaryotic ge-nome, Sc2.0 (www.syntheticyeast.org), and here we report the total synthesis of the first complete designer yeast chro-mosome.

Yeast chromosome III, the third smallest in S. cerevisiae (316,617 bp), containing the MAT locus determining mating type, was the first chromosome sequenced (12). We designed synIII according to fitness, genome stability and genetic flexibility principles devel-oped for the Sc2.0 genome (2). The native sequence was edited in silico using a series of deletion, insertion, and base substitution changes to produce the desired “designer” sequence (Figs. 1, S1, S2 and Supplementary Text). The hierarchical wet-lab workflow used to construct synIII (Fig. 2) consisted of three major steps: 1) The 750 bp “build-ing blocks” (BBs) were produced start-ing from overlapping 60- to 79-mer oligonucleotides and assembled using standard PCR methods (13, 14) by un-dergraduate students in the Build-A-Genome class at Johns Hopkins Uni-versity (Fig. 2A) (15). The arbitrary

Total Synthesis of a Functional Designer Eukaryotic Chromosome Narayana Annaluru,1* Héloïse Muller,1,2,3,4* Leslie A. Mitchell,2 Sivaprakash Ramalingam,1 Giovanni Stracquadanio,2,5 Sarah M. Richardson,5 Jessica S. Dymond,2,6 Zheng Kuang,2 Lisa Z. Scheifele,2,7 Eric M. Cooper,2 Yizhi Cai,2,8 Karen Zeller,2 Neta Agmon,2 Jeffrey S. Han,9 Michalis Hadjithomas,10 Jennifer Tullman,5 Katrina Caravelli,1 Kimberly Cirelli,1 Zheyuan Guo,1 Viktoriya London,1 Apurva Yeluru,1 Sindurathy Murugan,5 Karthikeyan Kandavelou,1,11 Nicolas Agier,12,13 Gilles Fischer,12,13 Kun Yang,2,5 J. Andrew Martin,2 Murat Bilgel,1 Pavlo Bohutski,1 Kristin M. Boulier,1 Brian J. Capaldo,1 Joy Chang,1 Kristie Charoen,1 Woo Jin Choi,1 Peter Deng,1 James E. DiCarlo,1 Judy Doong,1 Jessilyn Dunn,1 Jason I. Feinberg,1 Christopher Fernandez,1 Charlotte E. Floria,1 David Gladowski,1 Pasha Hadidi,1 Isabel Ishizuka,1 Javaneh Jabbari,1 Calvin Y. L. Lau,1 Pablo A. Lee,1 Sean Li,1 Denise Lin,1 Matthias E. Linder,1 Jonathan Ling,1 Jaime Liu,1 Jonathan Liu,1 Mariya London,1 Henry Ma,1 Jessica Mao,1 Jessica E. McDade,1 Alexandra McMillan,1 Aaron M. Moore,1 Won Chan Oh,1 Yu Ouyang,1 Ruchi Patel,1 Marina Paul,1 Laura C. Paulsen,1 Judy Qiu,1 Alex Rhee,1 Matthew G. Rubashkin,1 Ina Y. Soh,1 Nathaniel E. Sotuyo,1 Venkatesh Srinivas,1 Allison Suarez,1 Andy Wong,1 Remus Wong,1 Wei Rose Xie,1 Yijie Xu,1 Allen T. Yu,1 Romain Koszul,3,4 Joel S. Bader,2,5 Jef D. Boeke,2,10,14† Srinivasan Chandrasegaran1† 1Department of Environmental Health Sciences, Johns Hopkins University School of Public Health, Baltimore, MD 21205, USA. 2High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. 3Institut Pasteur, Group Spatial regulation of genomes, Department of Genomes Genetics, F-75015 Paris, France. 4CNRS, UMR 3525, F-75015 Paris, France. 5Department of Biomedical Engineering and Institute of Genetic Medicine, Whiting School of Engineering, JHU, Baltimore, MD 21218, USA. 6Johns Hopkins University Applied Physics Laboratory, Research and Exploratory Development Department, Biological Sciences, Laurel, MD 20723, USA. 7Department of Biology, Loyola University Maryland, Baltimore, MD 21210, USA. 8University of Edinburgh, Edinburgh, Scotland, UK. 9Carnegie Institution of Washington, Baltimore, MD 21218, USA. 10Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA. 11Pondicherry Biotech Private Limited, Pillaichavady, Puducherry 605014, India. 12Sorbonne Universités, UPMC, Univ Paris 06, UMR 7238, Génomique des Microorganismes, F-75005 Paris, France. 13CNRS, UMR7238, Génomique des Microorganismes, F-75005 Paris, France. 14NYU Langone Medical Center, New York, NY 10016, USA.

*These authors contributed equally to this work.

†Corresponding author. E-mail: boekej01@nyumc.org (J.D.B.); schandra@jhsph.edu (S.C.)

Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871–base pair designer eukaryotic chromosome, synIII, which is based on the 316,617–base pair native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, transfer RNAs, transposons, and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in a-mater derivatives resulting from loss of the MATĮ�DOOHOH�RQ�V\Q,,,��7KH�FRPSOHWH�GHVLJQ�DQG�V\QWKHVLV�RI�V\Q,,,�establishes S. cerevisiae as the basis for designer eukaryotic genome biology.

EMBARGOED UNTIL 2:00 PM US ET THURSDAY, 27 MARCH 2014

TEST

Automation  characterisation  platform  v1.0

C.  Hirst,  R.  Kitney,  G.  Baldwin

Sample  prep

Outgrowth  and  arrangement

Culture  maintenance,  sampling  and  measurement

� Cells  kept  at  similar  phase  of  growth◦ Ensure  high  quality  data◦ Ensures  cells  are  at  an  appropriate  

population  for  assay

� Growth  and  measurement  separated◦ Reduces  noise  in  data◦ Greatly  reduces  evaporation

Sample  prep

Outgrowth  and  arrangement

Culture  maintenance,  sampling  and  measurement

0Time  hours: 1 2 3 4 5 76

Flow  Cytometry

Flow  Cytometry

Induction

Dilution

8

Dilution

Automation  characterisation  platform  v1.0

C.  Hirst,  R.  Kitney,  G.  Baldwin

Anderson  22x  promoter  characterisation

0

0.5

1

1.5

2

2.5

3

Prom

oter  Outpu

t  (RP

U)

Promoter

Plate   reader

Flow  Cytometer

Pearson's coefficient  =  0.996

y  =  0.9699xR²   =  0.99292

0.01

0.1

1

100.01 0.1 1 10

Flow

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Plate   Reader   RPU

t =  180  min  

Single  cell  versus  population  measurements  show  high  degree  of  correlation  C.  Hirst,  R.  Kitney,  G.  Baldwin

Data  Sheets  for  Parts  and  SynBIS

Automation  characterisation  platform  v2.0  @Imperial  College

Summary• Automation  and  standardised metrology   is  accelerating    the  application  of  synthetic  biology

• Data  for  part  /  device  characterisation is  being  shared  openly

• New  chassis  are  being  constructed  e.g.  Sc2.0• Standards  are  being  developed  and  shared  • Huge  growth  and  interest  by  younger  researchers• Non-­‐biologists   are  now  doing  synthetic  biology  e.g.  Engineers  

• Significant  growth  of  community  labs  worldwide-­‐see  (www.biobuilder.org)