Algorithms For The Design And Assembly Of A Modular, Synthetic … · 2011. 9. 7. · Joel Bader & Jef Boeke High Throughput Biology Center, ... 12 Mb genome on 16 chromosomes 6000

Algorithms For The Design And Assembly Of A Modular, Synthetic Genome For Yeast

Sarah Richardson

Jessica Siegel Dymond

Joel Bader & Jef BoekeHigh Throughput Biology Center, Johns Hopkins University School of Medicine

20th Annual DOE CSGF ConferenceJuly 22 2011

Wednesday, July 27, 2011

Synthetic Biologythe design and fabrication of biological systems

Existing Systems

bacteriophage T7

Escherichia coli

Mycoplasma mycoides

Saccharomyces cerevisiae

New Systems

Control logicOscillators, Repressilators

Gene circuitryBioBricks

Systems biologyMetabolic Networks

DOIs: 10.1038/nature08753, 10.1186/1754-1611-4-1, 10.1038/nbt.1557, 10.1016/j.cell.2009.04.048, 10.1038/msb4100025, 10.1126/science.1126439, 10.1126/science.1190719Wednesday, July 27, 2011

Saccharomyces Cerevisiae 1.0a well characterized and beloved organism

small & highly amenable to manipulation - grows as haploid and diploid12 Mb genome on 16 chromosomes

6000 genes, ~1000 essential to the haploid cell, 250 introns“Generally Regarded as Safe” by the FDA

Pringle et al. J Bacteriol 140, 289–293 (1979)


Even a very “modest” change may have a negative impact on fitness - and the effects add up !

v0.0 v0.1 v0.2 v0.3 X

The Fitness Ratchet


loxPsym and Cre recombinaseloxPsym loxPsymgene

several arrangements can occur

induce Cre recombinasewith Estradiol



Cre several arrangements can occur




Cre several arrangements can occur




Cre Creseveral arrangements can occur




Cre Creseveral arrangements can occur



The Evolutionary Method

induce Cre recombinase

v0.0 v0.1 v0.2 v0.3 X

v1.0 v1.1 v1.2 v1.3 X

induce Cre recombinase

v2.0 v2.1 v2.2 v2.3 X

select for trait

select for trait


Saccharomyces Cerevisiae 2.0To study:

Genome structure and organization

Minimal genome sets

Genome evolution

Intron function and essentiality

Small RNA identification and classification

Our design will:Remove all introns, tRNA genes,

transposons, and associated repeats

“Tag” every gene for detection

Seed genome with site-specific recombination sites to foster

genomic rearrangements

Seed genome with restriction enzyme recognition sites to enable

modular assembly


peptide

gene

Computer Assisted Synthetic Design

www.genedesign.org


http://www.genedesign.org


peptide

gene

Computer Assisted Synthetic Design

www.genedesign.org

plasmid

chromosome

genome

BioStudio




Annotation

Visualization

Design

Version Control

Collaboration

Workflow Management

Analysis

OthersStudents Team Members

!!!

GFF3


Annotation - Sequence Ontology & GFF3


Visualization - GBrowse


Visualization - GBrowse


Design - PCR Tags

Pro: introduce unique “tags” in genomic featuresCon: may affect expression in unpredicted waysApp: PCRTags can be amplified to identify and locate synthetic sequence

ACTTCATTTACGAGCCAACCTTTGGAACCACTAAGCGATACTTCATTCTCATCACAACCACTGGAACCGTTGTCGGAT T S F S S Q P L E P L S DCDS

v1v2

PCRTag

Is it between 19 & 28 base pairs long?

Does it begin and end in a codon wobble?

Does the "most different" algorithm recode the sequence

by at least 33%?

Are the melting temperatures of both the original and recoded oligos between 58 and 60 ˚C?

Choose an exonic wild type oligo

Save oligos and choose again

YES

YES

YES

YES

NO

NO

NO

NO

Are both the original and recoded oligos unique in the

entire genome?

YES

NO


Design - PCR Tags!

!"!"#$

%&'()

!"!

%&'()

!

()

%&'

()

()

%&'

!"#$%&***+,-

()

%&'

WT

SYN

WT/ SYN

WT/ SYN

WT-SYN


Design - Codon Manipulation

* E Q I V R L L MDNA

M S F L N N P Q *ATGTCATTCCTGAATAACCCTCAGTAACAT

CDS 2

CDS 1

* E Q I V R L L M

M S F L N N P Q *ATGTCATTCCTGAATAACCCTCAGTAGCAT



T M G S Y G E T V DNA

M S F L N N P Q *ATGTCATTCCTGAATAACCCTCAGTAACAT

CDS 2

CDS 1

T M G S Y G E T A

M S F L N N P Q *ATGTCATTCCTGAATAACCCTCAGTAGCAT



Most Different (61%)

I W A N T S Y N T H E T I C G E N E Satctgggctaacacctcttacaacacgcacgaaactatttgcggtgaaaatgagagt

atctgggctaacacctcttacaacacCcacgaaacCatCtgTggtgaaaaCgaATCtatAtgggcAaaTacGAGCtaTaaTacCcaTgaGacGatAtgTggGgaGaaCgaATCAatTtgggctaacacTtcttacaacacAcacgaaacCatCtgcggtgaaaatgagTCA

Optimized (86%)

Least Different (86%)

ATCATTATA

TGG GCCGCGGCTGCA

ATCATTATA

GAGGAA

GAGGAA

GAGGAA

AACAAT

AACAAT

AACAAT

AGCTCCTCGTCTAGTTCA

AGCTCCTCGTCTAGTTCA

ACCACGACTACA

ACCACGACTACA

ACCACGACTACA

TACTAT

TGCTGT

GGCGGGGGTGGA

CACCAT

Most Different (61%)

I W A N T S Y N T H E T I C G E N E Satctgggctaacacctcttacaacacgcacgaaactatttgcggtgaaaatgagagt

atctgggctaacacctcttacaacacCcacgaaacCatCtgTggtgaaaaCgaATCtatAtgggcAaaTacGAGCtaTaaTacCcaTgaGacGatAtgTggGgaGaaCgaATCAatTtgggctaacacTtcttacaacacAcacgaaacCatCtgcggtgaaaatgagTCA

Optimized (86%)

Least Different (86%)

ATCATTATA

TGG GCCGCGGCTGCA

ATCATTATA

GAGGAA

GAGGAA

GAGGAA

AACAAT

AACAAT

AACAAT

AGCTCCTCGTCTAGTTCA

AGCTCCTCGTCTAGTTCA

ACCACGACTACA

ACCACGACTACA

ACCACGACTACA

TACTAT

TGCTGT

GGCGGGGGTGGA

CACCAT

PeptideOriginal


Design - Codon ManipulationBssSI

SRV SRA SRE SRD SRGPRV PRA PRE PRD PRGTRV TRA TRE TRD TRGARV ARA ARE ARD TRG

CAC GAGHE

nCA CGA Gnn

nnC ACG AGnATR ATS CTR CTS DTR DTS FTR FTS GTR GTS HTR HTS ITR ITS LTR LTS NTR NTS PTR PTS RTR RTS STR STS TTR TTS VTR VTS YTR YTS

CTC GTGLV

nCT CGT Gnn

nnC TCG TGnASC ASW CSC CSW DSC DSW FSC FSW GSC GSW HSC HSW ISC ISW LSC LSW NSC NSW PSC PSW RSC RSW SSC SSW TSC TSW VSC VSW YSC YSW

I W A N T S Y N T H E T I C G E N E S ORFDNA1 atctgggctaacacctcttacaacacccacgaaaccatctgtggtgaaaacgaatctDNA2 atctgggctaacacGAGttacaacacccacgaGaccatctgtggtgaaaacgaatct


yeast_chr9_3_27.A ~30 kb

yeast_chr9_3_27.A3 ~10 bp

yeast_chr08_3_29organism chromosome

numberchromosome

versiongenomeversion

yeast_chr9_0_00 ~300 kb

Version Control - Nomenclature

yeast_chr9_3_27.A3.11 ~750 bp

yeast_chr9_3_27.A3.11.o04 ~60 bp


Chromosome 9 (~230-1500kb)

in silico Chromosome Breakdown


A B C

Large Chunk 9.A(~30kb)




A B C


A1 A2 A3

Chunk 9.A2(~10kb)




A B C


A1 A2 A3

Chunk 9.A2(~10kb)

A201

A206

A212

Building Block 9.A2.06 (~750b)




A B C


A1 A2 A3

Chunk 9.A2(~10kb)

A201

A206

A212





A B C


A1 A2 A3

Chunk 9.A2(~10kb)

A201

A206

A212



CCAGGCCATCTCGATATGATCCATCTAGCGTTTCTCGACCAAGCAGCAGCTCTTATTCGTCAACGTGGGTCCGGTAGAGCTATACTAGG TTCGTCGTCGAGAATAAGCAGTTGTTC

5’ 3’

3’5’

Oligo 9.A2.06.o07 (~60b)



~60bp

~750bp

Chromosome Assembly12-18 Oligos

assembled by PCR

~80bp


~60bp

~750bp


assembled by PCR

~10kb

12-18 Building Blocks assembled with Gibson

Assembly

~80bp


~60bp

~750bp


assembled by PCR

~10kb


Assembly

~30kb

RsrII SfiI BstEII

Chunks assembled by restriction digestion and

ligation

~80bp


~60bp

~750bp


assembled by PCR

~10kb


Assembly

~30kb

RsrII SfiI BstEII

Chunks assembled by restriction digestion and

ligation

X

Chromosomes assembled by homologous recombination

~80bp


Restriction Enzyme Price and Placement

Chromosome Design



Chromosome Design



Cost(E) = ln Price(E)+A×max[0,|X −X0|

L−∆]+B0n0 +B1n1 +B2n2

1

Cost(E) = ln Price(E)+A×max[0,|X −X0|

L−∆]+B0n0 +B1n1 +B2n2

A = offset scoring constant∆ = offset allowance constantL = length of region

B0 = non-essential gene scoring constantn0 = number of non-essential genes modified

B1 = slow-growth gene scoring constantn1 = number of slow-growth genes modified

B2 = essential gene scoring constantn2 = number of essential genes modified

1

Chromosome Design


Large Chunk Boundaries(~30kb)

Chunk Boundaries(~10kb)

Mega Chunk Boundaries(~100kb)

Chromosome Design


m = number of enzymes to choose from n = number of boundary sites to choose




Chromosome Design



Brute force: O(mn)




Chromosome Design



Brute force: O(mn)




Nested, Overlapping Sub-Problems

Chromosome Design



Brute force: O(mn)Dynamic Programming: O(nm6)




Nested, Overlapping Sub-Problems

Chromosome Design


Time: 5 minutesAverage cost: 2.1

BioStudio:

Chromosome DesignTime: months of expert effortAverage cost: 7.6

Anonymous expert:


Build-A-Genome

>120 undergraduate and high school students since 200773% success rate and rapidly rising

>500,000 bp assembled from three chromosomes


Workflow Management - Database Schema


Results - LoxPsym Induction

WT

SYN

SYN


Results - Escape From The Fitness Ratchet


Results - Escape From The Fitness Ratchet

Fully synthetic chromosome arms function in yeast and generate phenotypic diversity by design

Jessica S. Dymond, Sarah M. Richardson, Candice E. Coombes, Timothy Babatz, Héloïse Müller,

Narayana Annaluru, William J. Blake, Joy Wu Schwerzmann, Junbiao Dai, Derek L. Lindstrom,

Annabel C. Boeke, Daniel E. Gottschling, Srinivasan Chandrasegaran, Joel S. Bader, Jef D. Boeke


AcknowledgementsJef Boeke

Jessica DymondEric Cooper

Lisa ScheifeleCandice E. Coombes

Timothy BabatzJunbiao Dai

Annabel C. Boeke

Joel BaderGiovanni StracquadanioYizhi CaiSindurathy Murugan

Srinivasan ChandrasegaranHéloïse MullerNarayana AnnaluruJoy Wu Schwerzmann

Krell StaffJeana GingeryMichelle King

Rachel Heidenreich

Build-A-GenomeJeffrey HanKatrina FoelberPablo Lee


Documents

Algorithms For The Design And Assembly Of A Modular, Synthetic … · 2011. 9. 7. · Joel Bader & Jef Boeke High Throughput Biology Center, ... 12 Mb genome on 16 chromosomes 6000