Reusable DNA Parts & Modular Assembly NICOLA J. PATRON Group Leader • Engineering Biology The Earlham Institute, Norwich, UK
We are a research institute tackling global challenges through life science research • 16 Research Groups • UK National Capability in Genomics • High Performance Computing Facility • >10 Tb new data per week
www.earlham.ac.uk
We are a research institute tackling global challenges through life science research • 16 Research Groups • UK National Capability in Genomics • High Performance Computing Facility • >10 Tb new data per week • Earlham Plant & Microbe DNA Foundry
www.earlham.ac.uk
181.5 million ha ‘biotech’ crops 18 million farmers
Single gene Transgenesis
“Modified” Organism
www.earlham.ac.uk
Small pathways
Large pathways
Synthetic Networks
HETEROLOGOUS TECHNOLOGIES Targeted addition MUTATIONAL TECHNOLOGIES Targeted Targeted Mutagenesis Recoding (editing)
Engineered “Chassis”
Single gene Transgenesis
“Modified” Organism
Small pathways
Large pathways
Synthetic Networks
TRANSGENIC TECHNOLOGIES Targeted addition MUTATIONAL TECHNOLOGIES Targeted Targeted Mutagenesis Recoding (editing)
Engineered “Chassis”
Automated assembly of complex and bespoke DNA molecules
www.earlham.ac.uk
Single gene Transgenesis
“Modified” Organism
Small pathways
Large pathways
Synthetic Networks
TRANSGENIC TECHNOLOGIES Targeted addition MUTATIONAL TECHNOLOGIES Targeted Targeted Mutagenesis Recoding (editing)
Engineered “Chassis”
Automated assembly of complex and bespoke DNA molecules Standardised DNA parts and assembly methods
www.earlham.ac.uk
Compatibility Interoperability Repeatability Quality Safety
Compatibility Interoperability Repeatability Quality Safety
1st Biological Standard: BioBrick BBF RFC10 Tom Knight, 2004
Y
Y
1st Biological Standard: BioBrick BBF RFC10 Tom Knight, 2004
Type IIS / Golden Gate Engler, Kandzia & Marillonet 2008
Y
Y
Phytobrick Standard for Plant Synthetic Biology: A Common Syntax for the Exchange of DNA Parts TSS 5'UTR
CODING SEQUENCE
5' NON TRANSCRIBED
GGAG
TGAC
DIST
Met
TACT
CORE
Met
CCAT(g)
5UTR
AATG
NTAG
Ala
AGCC Gly AGGT
CDS1
PROM + 5UTR A1
A2
POSITION
A3 NAME
B1
Ser
TTCG
CDS2
Stop
(*)GCTT
CTAG
B3
B4
FUNCTION
GGTA
3UTR
CGCT TERM
3UTR + TERM
CDS B2
PAS 3' NON TRANSCRIBED
TRANSCRIBED REGION
TCCC
PROX
3'UTR
B5
B6
C1
5' OVERHANG
3' OVERHANG
A1
DIST
Distal promoter region, cis regulator or transcriptional enhancer
GGAG
TGAC
A2
PROX
Proximal promoter region, cis regulator or transcriptional enhancer
TGAC
TCCC
A3
CORE
Minimal promoter region, including transcription start site (TSS)
TCCC
TACT
A4
5UTR
5ˈ untranslated region
TACT
CCAT
B2
NTAG
N terminal coding region
CCAT
AATG
B3
CDS1
Coding region - optional N terminal coding region
B4
CDS2
AGCC Patron et al. (2015) 208(1):13-9 AATG New Phytologist /AGGT Patron (2016) Biochemical Soc. Trans. 44(3), 702-708 AGCC - no start Coding region TTCG /AGGT or stop codon Rutten at al. (2015) BBF RFC106 RFC106
Phytobrick Standard for Plant Synthetic Biology: A Common Syntax for the Exchange of DNA Parts •
Characterised
Example DNA part data-sheet from Vazquez-Vilar et al (2017)
Phytobrick Standard for Plant Synthetic Biology: A Common Syntax for the Exchange of DNA Parts • •
Characterised Reusable
43521
T 56001
141414
T 43521
56002
141414 MarkYoules, Oleg Raitskin
Phytobrick Standard for Plant Synthetic Biology: A Common Syntax for the Exchange of DNA Parts • • •
Characterised Reusable Interoperable
GB3.0
Phytobrick Standard for Plant Synthetic Biology: A Common Syntax for the Exchange of DNA Parts • • • • •
Characterised Reusable Interoperable One-pot, one-step iterative assembly Unlimited size
UNIVERSITÄT HAMBURG • UNIVERSITY OF WESTERN SYDNEY • UNIVERSITY OF WARWICK • UNIVERSITY UNIVERSITY • UNIVERSITY OF WISCONSIN • COLORADO STATE UNIVERSITY • CINVESTAV • LANGFANG • ETH ZURICH • GYEONGSANG NATIONAL UNIVERSITY • UNIVERSITY OF CALIFORNIA, DAVIS • MURDOCH UNIVERSITY • INRA CENTRE DE RECHERCHE DE BORDEAUX • MAX PLANCK INSTITUTE FOR PLANT BREEDING RESEARCH • CHINESE ACADEMY OF TROPICAL AGRICULTURAL CHINESE ACADEMY OF SCIENCES • LEIBNIZ UNIVERSITAET HANNOVER • NEW YORK UNIVERSITY • UNIVERSITY OF LIVERPOOL • CENTRO DE BIOTECNOLOGÍA Y GENÓMICA WEST VIRGINIA UNIVERSITY AND SCHOOL OF MEDICINE • ACADEMY OF SCIENCES OF THE CZECH REPUBLIC • AUSTRALIAN NATIONAL UNIVERSITY • UNIVERSITY OF TENNESSEE • UNIVERSITY OF EDINBURGH • KAGAWA UNIVERSITY • ADAM MICKIEWICZ UNIVERSITY • IOWA STATE UNIVERSITY • UNIVERSITY OF YORK • TECHNISCHE UNIVERSITAET KAISERSLAUTERN • UNIVERSITAET ZU KOELN • FRIEDRICH-ALEXANDER-UNIVERSITAET • • INSTITUTE OF SCIENCE AND TECHNOLOGY AUSTRIA UNIVERSITY OF TARTU • INSTITUTE OF COTTON RESEARCH • COLD SPRING HARBOR LABORATORY • MARTIN-LUTHERUNIVERSITAET HALLE-WITTENBERG • PACIFIC NORTHWEST NATIONAL LABORATORY • UNIVERSITY OF MINNESOTA • OAK RIDGE NATIONAL LABORATORY • CSIRO • WAGENINGEN UNIVERSITY • USDA ARS • UNIVERSITY OF OSLO • UNIVERSITY OF EXETER • UNIVERSITY OF PENNSYLVANIA • INNER MONGOLIA UNIVERSITY • NEW YO
Toolkit requested by >200 institutes worldwide Sylvestre Marillonet
Engler et al (2014) ACS Synthetic Biology 3 (11), 839–843 http://www.addgene.org/cloning/MoClo/Patron/
DNA Foundries – Platforms for Automated Assembly, Delivery & Characterisation REGISTRIES
Part (Data)
Design Software
T T T T
T T T T
Part selection and construct design
Example DNA part data-sheet from Vazquez-Vilar et al (2017)
DNA Foundries – Platforms for Automated Assembly, Delivery & Characterisation REGISTRIES
Part (Data)
REPOSITORIES
Design Software
T T T T
T T T T
Part selection and construct design
Part (DNA)
Collect DNA parts from Repository and order synthesis of new parts
DNA Foundries – Platforms for Automated Assembly, Delivery & Characterisation REGISTRIES
Part (Data)
REPOSITORIES
BUILD
Design Software
T T T T
T T T T
Part selection and construct design
Part (DNA) Assembly
Collect DNA parts from Repository and order synthesis of new parts
Assembly and QA
• 384 simultaneous assembly reactions. Down to 125 nL volume with 1 nM DNA input. Transform into 1 µL E. coli cells. • Automated bacterial transformation, spreading and colony picking • HTP Validation by sequencing (Illumina or PacBio – ‘SMARTGATE’) – up to 10k samples simultaneously Construct with standard overhangs
BsaI
GGTCTCNNNNNNNN...NNNNNNGAGACC CCAGAGNNNNNNNN...NNNNNNCTCTGG BsaI
One-step TypeIIS-mediated addition of SMRTBells
NNNNNNNNNNNN NNNNNNNNNNNN
Polymerase and sub-reads provide a consensus read of complete insert
6
D’Amore et al (in review)
Part (Data)
DATA
REPOSITORIES
BUILD
Design Software
T T T T
DATA
REGISTRIES
T T T T
Part selection and construct design
Part (DNA) Part Data to Registry
Assembly
Collect DNA parts from Repository and order synthesis of new parts
Assembly and QA
Validation & Characterization Experiments
www.earlham.ac.uk
We use genomic and synthetic biology technologies to
We use genomic and synthetic biology Image: NJ Patron Image: A Giordano technologies to
• Engineer photosynthetic organisms for rapid, highyielding biosynthesis of high-value proteins and metabolites
• Engineer photosynthetic organisms for rapid, highyielding biosynthesis of high-value proteins and metabolites
• Increase the yield potential and nutritive value of crops
Image: Medicago Bioproduction Systems
Image: Raimond Spekking CC 4.0
Comparative genomics promoter architecture & cis-regulation of endogenous and heterologous regulatory elements
A Yaomin Cai
B
Controlling relative gene expression with tuneable, synthetic transcription factors and Boolean logic
Synthetic Gene Networks
“Modified” Organism
www.earlham.ac.uk
TRANSGENIC TECHNOLOGIES Targeted addition MUTATIONAL TECHNOLOGIES Targeted Targeted Mutagenesis Recoding (editing)
Engineered “Chassis”
Amanda Salzman
“Modified” Organism
TRANSGENIC TECHNOLOGIES Targeted addition
MUTATIONAL TECHNOLOGIES Targeted Targeted Mutagenesis Recoding (editing) CRISPR/Cas-mediated multiplexed targeted mutagenesis and gene stacking Oleg Raitskin
Lawrenson et al. (2015) www.earlham.ac.uk Parry et al (2015) Patron & Raitskin (2016)
Engineered “Chassis”
“Modified” Organism
TRANSGENIC TECHNOLOGIES Targeted addition
Engineered “Chassis”
MUTATIONAL TECHNOLOGIES Targeted Targeted Mutagenesis Recoding (editing) CRISPR/Cas-mediated Carbon flux multiplexed targeted lignin and mutagenesis and gene chlorogenic stacking CRISPR acid Oleg Raitskin Nathalia Volpi
www.earlham.ac.uk
?
in Engineering plants CROPS for improved bioproduction (O’Connor-JIC)
Single gene Transgenesis
“Modified” Organism
Small pathways
Large pathways
Synthetic Networks
TRANSGENIC TECHNOLOGIES Targeted addition MUTATIONAL TECHNOLOGIES Targeted Targeted Mutagenesis Recoding (editing)
Engineered “Chassis”
Automation specialist: DNA assembly, genotyping, sequencing, transfection Tony West
Automated assembly of complex and bespoke DNA molecules
Standardised, exchangeable DNA parts www.earlham.ac.uk
Yaomin Cai Oleg Raitskin Amanda Salzman Nathalia Volpi Tony West
Earlham Institute Anthony Hall Daniel Swan Liverpool GeneMill Imperial Foundry Edinburgh Foundry
www.earlham.ac.uk
The John Innes Centre Anne Osbourn Sarah O’Connor George Lomonossoff Wendy Harwood - Tom Lawrenson Brande Wulff - Asyraf Hatta Alison Smith - Aytug Tuncel
UCambridge Jim Haseloff - Bernardo Pollack PUC Chile Fernan Federici UCampinas Paulo Mazzafera
Emerging Leaders in Biotechnology for Public Good
IBMCP Diego Orazez IPB Halle Sylvestre Marillonet UEssex Christine Raines - Patty Lopez BB/P010490/1 BB/N019466/1 BB/L014130/1 BB/M000966/1