Introduction to Synthetic Biology423

2013Herbert Sauro

hsauro@u.washington.eduwww.sys-bio.org

Gene and Genomes

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Smallest Genome – was in 1999

One of the smallest Genomes: Mycoplasma genitalium (Small parasitic bacterium)

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Single Gene

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Smallest GenomeTotal genes: 521Protein coding genes: 482tRNA and rRNA: 39

This genome is of interest to synthetic biology because Craig Venter wants to use this organism as the basis for a minimal organism for genetic engineering.

Venter’s group has removed roughly 101 genes and the organism is still viable, the idea then is to patent the minimal set of genes required for life.

PNAS (2006) 103, 425--430

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Gene Function

The complexity of simplicityScott N Peterson and Claire M FraserGenome Biol. 2001;2(2):COMMENT 2002. Epub 2001 Feb 8.

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But the real prize goes to….

160-Kilobase Genome of the Bacterial Endosymbiont CarsonellaSymbiont of sap sucking PSYLLIDS or ‘jumping plant lice’ ~182 genes

The 160-Kilobase Genome of the Bacterial Endosymbiont Carsonella

Atsushi Nakabachi, Atsushi Yamashita, Hidehiro Toh, Hajime Ishikawa, Helen E. Dunbar, Nancy A. Moran, and Masahira Hattori

(13 October 2006)

Science 314 (5797), 267.

Endosymbiont : organism that livesin another cells.

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Prokaryotic Cells: E. coli

2-3 um

http://www.ucmp.berkeley.edu/bacteria/bacteriamm.html

1 .Bacteria lack membrane bound nuclei 2. DNA is circular3. No complex internal organelles

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Prokaryotic Cells: E. coli

http://atlas.arabslab.com

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Comparison to Eukaryotic Cells

http://www.cod.edu/people/faculty/fancher/ProkEuk.htm

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E. coli Cytoplasm

David S. Goodsell (Scripps)

Average spacing between proteins: 7 nm/molecule

Diameter of a protein: 5 nm

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E. Coli Statistics

David S. Goodsell (Scripps)

Length: 2 to 3 umDiameter: 1 umGeneration time: 20 to 30 mins

Translation rate: 40 aa/secTranscription rate: 70 nt/sec

Number of ribosomes per cell : 18,000

Small Molecules/Ions per cell:

Alanine: 350,000Pyruvate: 370,000ATP: 2,000,000Ca ions: 2,300,000Fe ions: 7,000,000

Data from: http://bionumbers.hms.harvard.eduhttp://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi

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E. Coli StatisticsE coli has approximately 4300 protein coding genes.

Protein abundance per cell:

ATP Dependent helicase: 104

LacI repressor: 10 to 50 moleculesLacZ (galactosidase) : 5000

CheA kinase (chemotaxis): 4,500CheB (Feedback): 240CheY (Motor signal): 8,200Chemoreceptors: 15,000

GlycolysisPhosphofructokinase: 1,550Pyruvate Kinase: 11,000Enolase: 55,800Phosphoglycerate kinase: 124,000

Krebs CycleMalate Dehydrogenase: 3,390Citrate Synthase: 1,360Aconitase: 1630Source: Protein abundance profiling of the Escherichia coli cytosol.

BMC Genomics 2008, 9:102. Ishihama et al.

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E. Coli StatisticsE coli has approximately 4300 protein coding genes.

Protein abundance per cell:

ATP Dependent helicase: 104

LacI repressor: 10 to 50 moleculesLacZ (galactosidase) : 5000

CheA kinase (chemotaxis): 4,500CheB (Feedback): 240CheY (Motor signal): 8,200Chemoreceptors: 15,000

GlycolysisPhosphofructokinase: 1,550Pyruvate Kinase: 11,000Enolase: 55,800Phosphoglycerate kinase: 124,000

Krebs CycleMalate Dehydrogenase: 3,390Citrate Synthase: 1,360Aconitase: 1630Source: Protein abundance profiling of the Escherichia coli cytosol.

BMC Genomics 2008, 9:102. Ishihama et al.

Molecules Numbers in Prokaryotes:

1. Ions Millions2. Small Molecules 10,000 – 100,0003. Metabolic Enzymes 1000 – 10,000s4. Signaling Proteins 100 – 1000s5. Transcription Factors 10s to 100s6. DNA 1 – 10s

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Circular Chromosome in E. coli

Most Prokaryotic DNA is circular. Gene arelocated on both strands of the DNA. Geneson the outside are transcribed clockwiseand those on the inside anticlockwise.

E. coli’s genome is 4,639,221 base pairs

Coding for 4472 genes, of which 4316are genes that code for proteins.

Proteins 4316

tRNAs 89

rRNAs 22

Other RNAs 64

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Circular Chromosome in E. coli

88% of the E. coli genome codes forproteins, the rest includes RNA coding,promoter, terminators etc.

In contrast, the Human genome:

3,000,000,000 base pairs andabout 25,000 genes.

Only 2% of the Human genome codesfor proteins. The rest is……RNA regulatory network? Human genes are also segmentedinto Exon and Introns, with alternative splicing,significantly increasing the actual number of protein

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EcoCyc: http://ecocyc.org/

E. coli Gene Structure

Page 134

Start codon

Stop codon (TAG, TAA, TGA)

RNA Polymerase Binds to Promoters

http://mgl.scripps.edu/people/goodsell/pdb/pdb40/pdb40_1.html

mRNAChanges in the promoter sequencecan change the efficiency of RNApolymerase binding to the DNA.

The promoter is therefore a sitewhich can be engineered.

Strong and Weak Promoters

TTGATA -- 16 -- TATAATTTGACA -- 17 -- TATAAT

Strong Promoter. The recA promoter is a strong promoter.

CTGACG -- 18 -- TACTGTTTGACA -- 17 -- TATAAT

Weak Promoter. The araBAD promoter is a weak promoter.

It differs from the averaged promoter sequence by one nucleotide and on base pair in the spacer region.

Most common Promoter (Consensus sequence)

The strength of a promote is one of the factors which determines the rate of transcription.

RNA Polymerase Stops at a Terminator

Changes in the terminator sequencecan change the efficiency of RNApolymerase stopping. If the gene is part of an operon, terminators can modulate relative expression levels of the different genes in the operon.

The terminator is therefore a sitewhich can be engineered.

Operon Structure

Gene A Gene B Gene C

TerminatorPromoter100% 60% 30%

Operators – Regulating Expression

Gene Regulationlac Operon

PromoterPromoter

Operator

Metabolic Enzyme (output)

Sugar in Medium Relative β-galactosidase

Glucose 1

Glucose + lactose 50

Lactose 2500

lacZ codes for β-galactosidase.lacY codes for β-galactoside permease.

Gene Regulationlac Operon

Lac repressor

Promoter Promoter

Operator

Metabolic Enzyme (output)

Gene Regulationlac Operon

LacI Repressor

lacI is a tetramer (x4)

LacI binding to Promoter

Ribosome Binding Sites

In summary:

Gene TerminatorRBSPromoter

Operators

Start Codon Stop Codon

5’-UTR

This course is about networks: The Science and Engineering of Biological Networks

The world is full of networks

WWW

SocialRoad

Electronic

Biological Networks

Metabolic NetworksMetabolic

About 1000-1400 genes that code formetabolic enzymes in E. coli (out of a totalof about 4300 genes)

Protein-Protein NetworksProtein Signaling Network

Protein-Protein NetworksProtein Signaling Network: CellDesigner

Kohn MIMS

20% of the human protein-coding genes encode components of signaling pathways, including transmembrane proteins, guanine-nucleotide binding proteins (G proteins), kinases, phosphatases and proteases.

Protein-Protein NetworksC

Genetic NetworksGene Regulatory Networks: BioTapestry

Genetic Networks

Gene Regulatory Networks: BioTapestry : Ventral Neural Tube in Vertebrate Embryo

Genetic Units

Understanding the Dynamic Behavior of Genetic Regulatory Networks by Functional Decomposition. William Longabaugh and Hamid Bolouri Curr Genomics. Author manuscript; available in PMC 2007 December 12. Published in final edited form as: Curr Genomics. 2006 November; 7(6): 333–341.

Hybrid Network: Cell Cycle Control is Bacteria

Two Kinds of Representations

1. Non-Stoichiometry – or ball and stick networksNo stoichiometry, kinetics or mass conservation

2. Stoichiometry – reaction maps

?? – Stuff that people make up, whose knows what they really mean

Cytoscape: Ball and Stick

Stoichiometric

Network Classification

Networks

Stoichiometric

Elementary

Non-Elementary

Non-Stoichiometric

Probabilistic

Ball and Stick (Data dependent)

Systems and Synthetic Biology

Systematic Biology

Synthetic Biology

Network Physiology

Systems Biology Synthetic Biology

Top Down

Bottom Up

Top Down and Bottom UpTop Down “-omics”

• Whole cellSystem

• Statistical CorrelationsModel

• High-throughputData Yeast Protein-Protein Interaction Map

Top Down and Bottom UpTop Down “-omics”

• Whole cellSystem

• Statistical CorrelationsModel

• High-throughputData

• Networks/PathwaysSystem

• Mechanistic, biophysicalModel

• Quantitative, single-cellData

Bottom Up ”mechanistic”