Post on 31-Jul-2015
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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
e
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
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® 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.
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