Reusable DNA parts and modular assembly- Nicola Patron

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