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Build Your Next Breakthrough Using Next-Generation CloningWednesday, February 27, 2013

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Presented by:

Marcus GrafDirector of Operations, Synthetic Biology,Life Technologies

Marcus Graf is the cofounder of GeneArt® and previously worked on the development of a DNA vaccine for HIV at the University of Regensburg. This DNA vaccine was one of the first proprietary and successfully produced and optimized constructs which led to the foundation of GeneArt®, today part of Life Technologies.

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Outline

Gene Synthesis Introduction and Overview

Multi-parameter Gene Optimization

Mammalian Expression Multigene Study

Process Overview and Production Times

Gene Online Ordering

GeneArt® Strings™ DNA Fragments

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What are GeneArt® Synthetic Genes?

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

Synthetic Genes can routinely be made >10kb in length

Genes are delivered in a GeneArt® standard cloning vector (pMX series) or the vector of the customer’s choice (additional shipable to the standard gene)

Standard deliverable is 5 µg lyophilized DNA, larger amounts are available based on additional plasmid preparation (additional shipable here as well)

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

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Key Applications for Synthetic Genes

Gene Synthesis

Gene Optimization

Basic Research

Functional assays

Antibodyoptimization

&production

Protein & Enzyme

optimization&

production

Positive controls

PCR, TaqMan, AmpliSeq and more

siRNA rescue &

functional assays

Cell line optimization

DNA engineering

Genetic engineering

Interaction studies

Transient expression

Protein engineering

Antibody optimization

Crystallization and structure analyis

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

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GeneArt® (used) Gene Synthesis capacity / month (kbp)

10 20 40 80 150300 500

800

1,600

2,500

> 4,000

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 End of 2010

0

1000

2000

3000

4000

5000

‘Synthia‘

592

Ecoli K12 4369

40%

35%

18%7%

% Non optimizedMammaliaBacteriaOther

Ordered base pairs gene synthesis Share of wild tpye vs. optimized

Capacity > 5.5 Mbp/monthGeneArt® Gene Synthesis

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Opportunities offered by Gene Synthesis

cost-effective, time- and resource-saving method for obtaining individual and desired DNA constructs with 100% sequence accuracy providing equivalent or better expression performance.

GeneArt® Gene Synthesis increases quality, reliability and success rate

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Coding Region (CDS)

codon usage

GC content

cryptic splice sites

direct repeats

RNA sec. structures

instability sequences

GeneOptimizer®

CDS

DNA

RNA

Polymerase

mRNAtRNA

rRNA

Amino

acids

Anti codon

Proteins

Polypeptide

chain

Ribosome

CodonmRNA

RNA

nucleotides

Gene Expression is influenced by many different factors

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GeneOptimizer®sliding window

C: Monte Carlo

A: Iterative

B: Test all possible

How to solve the multi-parameter optimization problem?

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GeneOptimizer® pat. pend.

wild typesequence

CDS

Geneticstability

TranscriptionProcess

Stabilityof mRNA

TranslationProcess

Further individual requirements, e.g. specific cloning sites and/or motifs&target vector determinations

&

More than 10 years of gene optimization experienceSolved by GeneOptimizer®

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Multigene study in Mammalian HEK293T Cells

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

Protein Kinases

Transcription Factors

Membrane Proteins

Ribosomal Proteins

Cytokines

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Jnk3

40

optimized

Mo

ck

PP

1

PP

2

PP

3

PP

1

PP

2

PP

3

wildtype

40 CREB1

AQP5

60

30

IL-2

15

20

SMARCD140

Kinases

Transcription

Factors

Membrane

Proteins

Cytokines

Ribosomal

Proteins

( 12 ) ( 12 ) optopt > > wtwt

wtwt = = optopt ( 3 )( 3 )

( 4 ) ( 4 ) optopt > > wtwt

( 16 ) ( 16 ) optopt > > wtwt

wtwt = = optopt ( 1 )( 1 )wtwt >> optopt ( 1 )( 1 )

( 7 ) ( 7 ) optopt > > wtwt

wtwt = = optopt ( 1 )( 1 )

( 4 ) ( 4 ) optopt > > wtwt

(B)(A) (C)

▲

▲

▲

▲

▲

wildtype optimized

wildtype optimized

wildtype optimized

wildtype optimized

wildtype optimized

Jnk3

CREB1

AQP5

IL-2

SMARCD1

x 2.2

x 13

x 9

absolut

x 1.3re

lati

ve

rela

tiv

e

exp

ressio

nexp

ressio

nre

lati

ve

rela

tiv

e

exp

ressio

nexp

ressio

nre

lati

ve

rela

tiv

e

exp

ressio

nexp

ressio

nre

lati

ve

rela

tiv

e

exp

ressio

nexp

ressio

nre

lati

ve

rela

tiv

e

exp

ressio

nexp

ressio

n

Jnk3

40

optimized

Mo

ck

PP

1

PP

2

PP

3

PP

1

PP

2

PP

3

wildtype

40 CREB1

AQP5

60

30

IL-2

15

20

SMARCD140

Kinases

Transcription

Factors

Membrane

Proteins

Cytokines

Ribosomal

Proteins

( 12 ) ( 12 ) optopt > > wtwt

wtwt = = optopt ( 3 )( 3 )

( 4 ) ( 4 ) optopt > > wtwt

( 16 ) ( 16 ) optopt > > wtwt

wtwt = = optopt ( 1 )( 1 )wtwt >> optopt ( 1 )( 1 )

( 7 ) ( 7 ) optopt > > wtwt

wtwt = = optopt ( 1 )( 1 )

( 4 ) ( 4 ) optopt > > wtwt

(B)(A) (C)

▲

▲

▲

▲

▲

wildtype optimized

wildtype optimized

wildtype optimized

wildtype optimized

wildtype optimized

Jnk3

CREB1

AQP5

IL-2

SMARCD1

x 2.2

x 13

x 9

absolut

x 1.3

Jnk3

40

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

40 CREB1

AQP5

60

30

IL-2

15

20

SMARCD140

Kinases

Transcription

Factors

Membrane

Proteins

Cytokines

Ribosomal

Proteins

( 12 ) ( 12 ) optopt > > wtwt

wtwt = = optopt ( 3 )( 3 )

( 4 ) ( 4 ) optopt > > wtwt

( 16 ) ( 16 ) optopt > > wtwt

wtwt = = optopt ( 1 )( 1 )wtwt >> optopt ( 1 )( 1 )

( 7 ) ( 7 ) optopt > > wtwt

wtwt = = optopt ( 1 )( 1 )

( 4 ) ( 4 ) optopt > > wtwt

(B)(A) (C)

▲

▲

▲

▲

▲

wildtype optimized

wildtype optimized

wildtype optimized

wildtype optimized

wildtype optimized

Jnk3

CREB1

AQP5

IL-2

SMARCD1

x 2.2

x 13

x 9

absolut

x 1.3re

lati

ve

rela

tiv

e

exp

ressio

nexp

ressio

nre

lati

ve

rela

tiv

e

exp

ressio

nexp

ressio

nre

lati

ve

rela

tiv

e

exp

ressio

nexp

ressio

nre

lati

ve

rela

tiv

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exp

ressio

nexp

ressio

nre

lati

ve

rela

tiv

e

exp

ressio

nexp

ressio

n

wildtypewildtype > > optimizedoptimized ( 1 ) ( 1 )

optimizedoptimized > > wildtypewildtype ( 44) ( 44)

wildtypewildtype = = optimizedoptimized ( 5 ) ( 5 ) wildtypewildtype > > optimizedoptimized ( 1 ) ( 1 )

optimizedoptimized > > wildtypewildtype ( 44) ( 44)

wildtypewildtype = = optimizedoptimized ( 5 ) ( 5 )

Yields: 88% of proteins express higher with optimized genes

Reliability: 100% of optimized genes were expressed while 12% of wildtype genes showed no detectable expression

Fath et al. (2011)A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression. PLoS ONE 6(3): e17doi:10.1371/journal.pone.0017596

Multiparameter RNA and Codon Optimization

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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

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3 fold relative expression

after GeneOptimizer® optimization

compared to wild type protein

Comparative expression analysis of wild type versus an optimized human gene:

(A) Western Blot analysis using α-His antibodies. Expression levels of 2 independent transfections per wildtype and optimized construct are displayed. Standardization is based on endogenous α-His protein.

(B) Summary: Relative expression levels of wildtype versus optimized constructs of three independent transfections are displayed

GeneOptimizer® success story continuedAnalysis for mammalian expression

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Traditional cloning vs. GeneArt® Gene synthesis: value

Order Oligos

PCR reaction and purification

Cloning Kit

PCR screening

Midi prep

Agarose gel analysis

Sequencing

A typical 330 amino acid protein / 1000 base pair gene

Order online (Design & Optimize)

Production

Shipment (100% sequence verified)

Traditional Cloning

Time 4 hours (á $35)

= $140.00

Material + $152.20

Failure rate (+20%) + $58.45

Total Cost = $350.65

in 8-10 days

Expression rate 1x wild type

GeneArt® Gene Synthesis

Time 20 minutes

= $8.33

Material + $350 (list price)

Failure rate (+20%) + $0

Total Cost = $358.33

in 10 days

Expression rate 3 -100x wild type

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Technology and Process Overview

Optimization/CAD/Portal

Design fragments and oligos

Alignment and simulation in silico

Nano scale synthesis

High throughput/quality

Paralleled approaches

High throughput

High throughput

Automated

Automated CE sequencing

Automated alignment

Product report: QAD

ISO 9001:2008

LIM

S

Hig

h T

hro

up

ut P

roce

ss

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Gene Length Processing time (business days)

Standard 1) Express 1) SuperSpeed 3)

≤1,200 bp 2) 9 7 5 (SuperSPEED 1.2)

1,201 2) - 1,800 bp 14 12 7 (SuperSPEED 1.8)

1,801 - 3,000 bp 14 12 n/a

3,001 - 5,000 bp 19 17 n/a

> 5,000 bp Get a quote Get a quote n/a

For complex sequences: Get a quote

1) Valid for standard gene synthesis (non-complex, GC content 10% - 80%).

2) In case of GC content of 10% - 20% or 75% - 80%: 1,000 bp instead of 1,200 bp.

3) Subject to sequence assessment. Order must be placed by 3:00 p.m. CET.

Please note: Rarely, requested sequences are found to be toxic and/or genetically unstable. These production times are only valid for nontoxic sequences that are genetically stable in E. coli.

GeneArt® Gene Synthesis Production Times

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SuperSPEED

De novo gene synthesis of sequences up to 1,2 kb in 5 business daysup to 1,8 kb in 7 business days

Emergency genes e.g. H1N1 in 2009: 1,8 kb in 4 days

Fast Access to any SequenceGeneArt® Gene Synthesis

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Excel® fileorEmail

Portal

Cart / Order

Manufacturing

Shipping

Scientist

Email

Customer Support

QuoteSupport

Vector NTI(launch Q2)

How to Order GeneArt® Serviceswww.lifetechnologies.com/geneartgenesynthesis

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Gene Synthesis

and Optimization

Subcloning*

Mutagenesis

Strings™ DNAFragments

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® ServicesGene Synthesis to Protein Expression

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GeneArt® Strings™ DNA FragmentsYour source for genes. Fast and affordable for every lab.

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GeneArt® Synthetic Gene Service and GeneArt® StringsTM

Ready to use: Sequence of

interest, de novo

synthesized and

cloned in

GeneArt®

standard vector

(pMX series).

100% sequence

accuracy,

confirmed by

QAD, quantity 5

µg plasmid DNA

Ready for cloning: Linear dsDNA

fragments up to 1,000

bp, de novo

synthesized and bulk

sequenced

quantity >200 ng

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GeneOptimizer® pat. pend.

wild typesequence

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

www.lifetechnologies.com/genesynthesis

GeneArt® Gene Synthesis Benefits

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For research use only. Not for use in diagnostic procedures.

© 2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. Excel is a registered trademark of Microsoft Corporation.

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