1 The world leader in serving science

XXX xxxx

Biosciences, Life Sciences Solutions,

Geneart AG, Regensburg, Germany

An introduction to Gene Synthesis –

capabilities and uses

2

Agenda

• Introduction in writing DNA and gene synthesis

• GeneArt® gene synthesis: characteristics, process and applications

• Gene optimization

• Economic gene synthesis: GeneArt® Strings DNA fragments

• Summary: GeneArt® gene synthesis benefits

3

Breeding

Accelerated

breeding

Biotechnology

colchizine

The biotechnological evolution

4

1975 1980 1985 1990 1995 2000 2005 2010

1 Tbp

1 Gbp

1 Mbp

1 kbp

automated dye terminator sequencing

manual radioactive sequencing

next generation sequencing

Exponential sequence data growth

5

automated dye terminator sequencing

manual radioactive sequencing

next generation sequencing

ha

rd d

isk c

ap

acity (

GB

) Exponential sequence data growth ... follows Moore’s Law

6

This is a vast amount of data

7

ATG ATC TGT CAC GCA GAG CTA

...which we

can read

...copy &

paste

...and just learn

to rewrite

This is a vast amount of data

compare thee to a summer ?Shall I day's

8

10

100

1.000

10.000

100.000

1.000.000

10.000.000

100.000.000

1965 1975 1985 1995 2005 2015

1.1

Mb

Gib

so

n: M

. m

yco

ide

s

33

bp

Ko

este

r: a

ng

iote

nsin

II

2.1

kb

Yo

un

g:

pla

sm

id

51

4 b

p E

dg

e: le

uko

cyte

inte

rfe

ron

41

bp

Ita

ku

ra: so

ma

tosta

tin

32

kb

Ko

du

ma

l: p

oly

ke

tid

e s

yn

tha

se

7.5

kb

Cello

: p

olio

vir

us

2.7

kb

Ste

mm

er:

pla

sm

id

77

bp

Ag

arw

al: a

la tR

NA

12

Mb

S

c 2

.0

invention of PCR

introduction of commercial

gene synthesis

1970 1975 1980 1985 1990 1995 2000 2005 2010 2015

100,000,000

10,000,000

1,000,000

100,000

10,000

1,000

100

10

pu

bli

ch

ed

co

ns

tru

ct

siz

e [

bp

]

27

3 k

b A

nn

alu

ru: S

c2

.0 s

yn

III

History of Writing DNA

9

1999: Foundation → GeneArt GmbH 3 employees

2006: Going public → GeneArt AG 60 employees

2011: GeneArt a part of... 200 employees (global 11,000)

2014: Life Technologies a brand of... 270 employees (global 50,000)

1999

Division: Synthetic BiologyDivision: Synthetic Biology

today

0.5 genes / month

> 6000 genes / month

The history of GeneArt® gene synthesis

10

Traditional Cloning Gene Synthesis

Availability DNA/RNA template needed

Safety Source organisms potentially harmful

Flexibility Sequence modifications are limited

Speed Varying success rate - tedious process

Origin Frequently uncertain origin of material

Traditional cloning

11

Traditional Cloning Gene Synthesis

Availability DNA/RNA template needed No template needed - just in silico data

Safety Source organisms potentially harmful No critical organisms involved - all in silico

Flexibility Sequence modifications are limited No design limitations - expression optimization

Speed Varying success rate - tedious process Starting at 5 days production time

Origin Frequently uncertain origin of material Certified origin and clear documentation

Save time and focus on your research

Traditional cloning vs. GeneArt® gene synthesis

12

• Synthetic Genes are double stranded DNA constructs, synthesized to the customer specification based on customers digital sequence

• Synthetic Genes can routinely be made > 10 kb in length

• Genes are delivered in a GeneArt® standard cloning vector or the vector of the customer’s choice

• Standard deliverable is 5 µg lyophilized DNA, larger amounts are available based on additional plasmid preparation

• All genes are 100 % sequence verified prior to shipment and come with quality assurance documentation

What are synthetic genes?

13

ATGAGTAAAGGA GAAGAACTTTTC ACTGGAGTTGTC CCAATTCTTGTT GAATTAGATGGC GATGTTAATGGG

ATGAG AA GG GA GA CT TTC ACTGG GTTGT CC ATTCT GT GA GA GGC GA GT AA GG

ATGAGCAAGGGC GAGGAGCTGTTC ACTGGCGTTGTG CCCATTCTGGTG GAGCTGGACGGC GACGTGAACGGC

How gene synthesis works

14

ATGAGCAAGGGCGAGGAG

ATGAGCAAGGGCGAGGAG

ATGAGCAAGGGCGAGGAG

A C G T

How gene synthesis works

15

How gene synthesis works

16

How gene synthesis works

17

colony

How gene synthesis works

18

How gene synthesis works

19

Gene Synthesis and

Optimization

Subcloning*

Mutagenesis

Strings™ DNA Fragments

Precision TALs

Plasmid preparation

Protein production

Cell line development

Comprehensive product and service portfolio

All from one hand

All in house production

Highest quality standards ISO 9001:2008 certified

Easy to order

* access to all LT cloning vectors!

GeneArt® Services – from Gene Synthesis to Protein Expression

20

Gene Synthesis

Gene Optimization

Basic Research

Functional assays

Antibody optimization

& production

Protein & Enzyme

optimization &

production

Positive controls

PCR, TaqMan®,

Ion AmpliSeq

siRNA rescue &

functional assays

Cell line optimization

DNA engineering

Genetic engineering

Interaction studies

Transient expression

Protein engineering

Antibody optimization

Crystallization and structure analysis

Immunogene optimization for DNA- and RNA vaccines

Gene therapy

Rescue of siRNA mediated knock-out

Host engineering

Cell line development

Stable protein expression for novel cellular screening assays

Drug and target validation

Key applications for synthetic genes

21

www.lifetechnologies.com

22

The Project Configurator

23

Open a gene synthesis service form

24

Order a gene synthesis

25

Editing & annotating

26

Optimising your sequence

27

While we wait... ...let‘s have a quick look „under the hood“ on the algorithm

Millions of codon combinations are evaluated...

28

Pagothenia

borchgrevinki

Homo

sapiens

Aequorea

victoria

Escherichia

coli

Arabidopsis

thaliana

In silico gene optimization

29

Pagothenia

borchgrevinki

Homo

sapiens

Aequorea

victoria

Escherichia

coli

Arabidopsis

thaliana

In silico gene optimization ... is based on the universal code

30

CDS

• Codon usage

• Overall GC content

• Restriction sites in&out

• Repetitive sequences

• RNA secondary structures

• mRNA halflife

• Ribosome entry sites

• Cryptic splice sites

• Premature polyA motifs

• Others ...

Computational multi-parameter

optimization

electronic sequence (DNA or protein)

optimized sequence

In silico gene optimization - The Gene Optimizer®

31

Sequence optimization enhances performance and expression of your genes

• Optimal codon quality for your host • Stabilized mRNA • Avoid unwanted motifs and secondary structures • Reduce sequence complexity (high or low GC-content, repetitions)

Wildtype sequence

Optimized sequence

Gene optimization

32

3’-UTR 5’-UTR

CTG

CTC

CTT

TTG

CTA

TTA

L

ATC

ATT

ATA

I

TTC

TTT

F

GAG

GAA

E ACC

ACA

ACT

ACG

T

GAG

GAA

E TGC

TGT

C

CAC

CAT

H

L

CTG

Amino Acid

Codon

Codon Quality

• Wild type not optimal for expression

• Best codon back translation not optimal w.r.t. unwanted motifs/repetitions/secondary structures

• Goal: find a tradeoff

Gene expression is influenced by many different factors

Considerations for sequence optimisation

33

Sliding window

Optimized

5’-UTR

Extended window

5’-UTR

5’-UTR

1st step

2nd step

6th step

The sliding window moves from 5’-UTR to 3’-UTR, one codon per step

“All possible” codon combinations (with CAI higher than a threshold) are tested

The extended window is considered for evaluating the codon combination

Only the first codon of the best combination is fixed

Up to three phases with more and more relaxed thresholds

The patented sliding window algorithm

34

After the (optional) optimisation the summary tab provides an overview of

the resulting sequence and its properties, such as GC content and codon

usage.

The summary tab

35

Gene optimization as a general strategy to improve autologous as well as

heterologous expression of human genes

50 (mammalia) or 100 (E.coli) standard human genes representing the most interesting

protein classes were selected from the NCBI data bank

Protein

Kinases

Transcription

Factors

Membrane

Proteins

Ribosomal

Proteins

Cytokines

Gene optimization as a general strategy to improve autologous expression of human genes

50 standard human genes representing the most interesting protein classes were selected from the NCBI data bank

Fath et al., 2012, PLoS ONE

Performance of gene optimization

36

Membrane Membrane

ProteinsProteins

Membrane Membrane

ProteinsProteins

TranscriptionTranscription

FactorsFactors

RibosomalRibosomal

& & otherother

ProteinsProteins

RibosomalRibosomal

& & otherother

ProteinsProteins

CytokinesCytokinesCytokinesCytokines

Protein Protein

KinasesKinases

Protein Protein

KinasesKinases

optimized

mo

ck

PP

1

PP

2

PP

3

PP

1

PP

2

PP

3

wildtype

wildtype optimized

JNK340

rela

tive

exp

ressio

n

60

35 CREB1

wildtype optimized

rela

tive

exp

ressio

n

60

SMARCD140

wildtype optimized

rela

tive

exp

ressio

n

60

AQP5

60

30

wildtype optimized

rela

tive

exp

ressio

n

IL-215

20

wildtype optimized

rela

tive

exp

ressio

n

opt > wt opt = wt opt < wt only opt

4 none none none

opt > wt opt = wt opt < wt only opt

4 none none none

opt > wt opt = wt opt < wt only opt

4 none none none

opt > wt opt = wt opt < wt only opt

4 none none none

opt > wt opt = wt opt < wt only opt

13 3 none none

opt > wt opt = wt opt < wt only opt

13 3 none none

opt > wt opt = wt opt < wt only opt

15 1 2 4

opt > wt opt = wt opt < wt only opt

15 1 2 4

opt > wt opt = wt opt < wt only opt

6 2 none 2

opt > wt opt = wt opt < wt only opt

6 2 none 2

▲JNK3

x 14

▲CREB1

x 2.8

▲SMARCD1

x 1.8

▲AQP5

x 9

▲IL-2

only opt

Fath et al., 2012, PLoS ONE

Mammalian expression: wildtype vs. optimized

37

opt > wt opt = wt opt < wt

opt > wt

86 %

opt = wt

10 %

opt < wt

4 %

• All gene-optimized constructs are expressed while expression of 12 % of wildtype genes was not detectable.

• 96 % of optimized genes display equal or better expression yield than their wildtype counterparts.

• Up to 25-fold increase in protein expression through optimization.

average variations ≤ 10% are considered equal (opt = wt) Fath et al., 2012, PLoS ONE

Mammalian expression: wildtype vs. optimized

38

JNK140

optimized

mo

ck

PP

1

PP

2

PP

3

PP

1

PP

2

PP

3

wildtype optimized

mo

ck

PP

1

PP

2

PP

3

PP

1

PP

2

PP

3

wildtype

rela

tive e

xp

ressio

n

wildtype optimized

+x 2.8

JNK160

60

40

JNK3

60

40 p38a

rela

tive e

xp

ressio

n

wildtype optimized

+x 25

JNK3

rela

tive e

xp

ressio

n

wildtype optimized

+x 3.1

p38a

▲

▲

▲

JNK140

optimized

mo

ck

PP

1

PP

2

PP

3

PP

1

PP

2

PP

3

wildtype optimized

mo

ck

PP

1

PP

2

PP

3

PP

1

PP

2

PP

3

wildtype

rela

tive e

xp

ressio

n

wildtype optimized

+x 2.8

JNK160

60

40

JNK3

60

40 p38a

rela

tive e

xp

ressio

n

wildtype optimized

+x 25

JNK3

rela

tive e

xp

ressio

n

wildtype optimized

+x 3.1

p38a

▲

▲

▲

NiNi--purificationpurificationmockmock, , wtwt, , optopt

ElutionElutionHisHis--taggedtagged proteinsproteins

pulldownpulldownSubstrate Protein Substrate Protein

BeadsBeads

Kinase Kinase assayassay(+ATP)(+ATP)

LysisLysisTriplicateTriplicate transfectiontransfection

mockmock, , wtwt, , optopt

Western Western BlotBlot

NiNi--purificationpurificationmockmock, , wtwt, , optopt

ElutionElutionHisHis--taggedtagged proteinsproteins

pulldownpulldownSubstrate Protein Substrate Protein

BeadsBeads

Kinase Kinase assayassay(+ATP)(+ATP)

LysisLysisTriplicateTriplicate transfectiontransfection

mockmock, , wtwt, , optopt

Western Western BlotBlot

JNK140

30

optimized

Mo

ck

PP

1

PP

2

PP

3

PP

1

PP

2

PP

3

wildtype

rela

tive e

xp

ressio

n

wildtype optimized

JNK1

x 2.8

JNK1

mo

ck

JN

K-w

t

JN

K-o

pt

70 -

55 -

35 -

GST-c-Jun

beads

- -Ser63P

GST-c-Jun(1-89)

mock wildtype optimized

rela

tive a

mo

un

t

ph

osp

ho

ryla

ted

su

bstr

ate

in vitro activity

mock wildtype optimizedre

lati

ve a

mo

un

t

JN

K1

JNK1 (recombinant)

mo

ck

JN

K-w

t

JN

K-o

pt

GST-c-Jun

beads

-Penta-His

JNK1

70 -

55 -

35 -

▲

Increase of expression yields does not affect solubility or functionality

In vitro activity

39

CDC235

60

optimized

mo

ck

PP

1

PP

2

PP

3

PP

1

PP

2

PP

3

wildtype

rela

tive e

xp

ressio

n

wildtype optimized

CDC2

x x 2.9

untransfected CDC2 siRNA only

CDC2 (optimized) + CDC2 siRNA CDC2 (optimized) + nonsilencing siRNA

+▲CDC235

60

optimized

mo

ck

PP

1

PP

2

PP

3

PP

1

PP

2

PP

3

wildtype

rela

tive e

xp

ressio

n

wildtype optimized

CDC2

x x 2.9

untransfected CDC2 siRNA only

CDC2 (optimized) + CDC2 siRNA CDC2 (optimized) + nonsilencing siRNA

+▲

Optimized synthetic genes represent valuable tools for functional genomics (RNAi)

Functional Genomics (RNAi)

40

Summary of study

Gene Design and Commercial Gene Synthesis ...

increases overall expression success rate

improves expression yields in general

does not alter activity of encoded protein

allows cost effective and quick availability

allows to confirm siRNA phenotypes

… is an Excellent Tool for Functional Genomics

41

Gene optimization as a general strategy to improve autologous as well as

heterologous expression of human genes

50 (mammalia) or 100 (E.coli) standard human genes representing the most interesting

protein classes were selected from the NCBI data bank

Protein

Kinases

Transcription

Factors

Membrane

Proteins

Ribosomal

Proteins

Cytokines

ATG ATC TGT CAC GCA GAG CTA

»Writ

ing DNA

ATGGCTGG....CGGTGC

Complete service chain: from gene to protein

42

GeneArt® Gene Synthesis

GeneArt® StringsTM

DNA Fragments

Cloning

Oligo assembly

Oligo synthesis

Bioinformatics

Final quality control

Fragment amplification

Screening

Sequencing

DNA preparation

Size: 824 861 274 533 577 2704 2630 2680 2663 bp

0.1 - 1.0 kb 1.0 - 3.0 kb

GeneArt® Strings™: The economic version of gene synthesis

43

GeneArt® StringsTM

DNA Fragments

Size: 824 861 274 533 577 2704 2630 2680 2663 bp

0.1 - 1.0 kb 1.0 - 3.0 kb

GeneArt® Strings™: The economic version of gene synthesis

44

Strings™ 0.1 - 1.0 kb

Oligo assembly

Oligo synthesis errors

Mutation

Enzymatic error correction

Strings™ 1.0 - 3.0 kb

Enzymatic error correction

... allows to get even larger

GeneArt® Strings™: Going beyond 1 kb

45

simple oligo assembly

with error correction

The effect of error correction on String™ synthesis

p=

0.9

8

46

Question Answer

What are GeneArt® Strings™?

• Synthetic linear, double stranded DNA pool from 0.1 to 3.0 kb length • Technology based on gene synthesis • Customer needs to specify 5‘ & 3‘ ends supporting cloning or assembly to larger genes

What is the production time of Strings™?

• 0.1 - 1.0 kb 5 business days • 1.0 - 3.0 kb 8 business days

What are the deliverables?

• > 200 ng dried PCR fragment DNA - ready for cloning

What quality control is included?

• Every String™ is checked by gel electrophoresis • Yield is quantified by OD260

• Bulk sequencing ensures customer sequence is highly represented in DNA fragment pool

GeneArt® Strings™ DNA Fragments at a glance

47

Alternative Applications of GeneArt® Strings™

In vitro translation Control template in quantitative real time PCR

Homologous recombination

in yeast

DNA hybridization

StringsTM DNA Fragments

Your favorite application

48

GeneOptimizer® pat. pend.

wild type sequence

Efficiency – De novo synthesis is cost effective and fast

Availability – all sequences are accessible, easy to order

Flexibility – no restrictions in design, no natural template is required

Performance – optimization significantly enhances the expression probability

Reliability – GeneArt® technology provides reliable delivery and success rates

Service offering – comprehensive portfolio from GeneArt® Strings™ to proteins

Summary: GeneArt® Gene Synthesis Benefits

49

Acknowledgements

Synthetic

Biology

R&D Team

Carlsbad

Synthetic

Biology

R&D Team

Regensburg

Synthetic Biology

Software Team

Singapore

MIT - Collaboration

Dept Biological Engineering

Chris Voigt, Ron Weiss

50

© 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc. used under permission and license.

51