Introduction to Synthetic Biology 423

1. Introduction to Synthetic Biology423 2013 Herbert Sauro hsauro@u.washington.edu www.sys-bio.org

2. Gene and Genomes

3. Smallest Genome – was in 1999 Single Gene } One of the smallest Genomes: Mycoplasmagenitalium (Small parasitic bacterium)

4. Smallest Genome Total genes: 521 Protein coding genes: 482 tRNA 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

5. Gene Function The complexity of simplicity Scott N Peterson and Claire M Fraser Genome Biol. 2001;2(2):COMMENT 2002. Epub 2001 Feb 8.

6. But the real prize goes to…. The 160-Kilobase Genome of the Bacterial EndosymbiontCarsonella Atsushi Nakabachi, Atsushi Yamashita, HidehiroToh, Hajime Ishikawa, Helen E. Dunbar, Nancy A. Moran, and Masahira Hattori (13 October 2006) Science314 (5797), 267. Endosymbiont : organism that lives in another cells. 160-Kilobase Genome of the Bacterial EndosymbiontCarsonella Symbiont of sap sucking PSYLLIDS or ‘jumping plant lice’ ~182 genes

7. Prokaryotic Cells: E. coli 1 .Bacteria lack membrane bound nuclei 2. DNA is circular 3. No complex internal organelles 2-3 um http://www.ucmp.berkeley.edu/bacteria/bacteriamm.html

8. Prokaryotic Cells: E. coli http://atlas.arabslab.com

9. Comparison to Eukaryotic Cells http://www.cod.edu/people/faculty/fancher/ProkEuk.htm

10. E. coli Cytoplasm  Average spacing between proteins: 7 nm/molecule Diameter of a protein: 5 nm David S. Goodsell (Scripps)

11. E. Coli Statistics Length: 2 to 3 um Diameter: 1 um Generation time: 20 to 30 mins Translation rate: 40 aa/sec Transcription rate: 70 nt/sec Number of ribosomes per cell : 18,000 Small Molecules/Ions per cell: Alanine: 350,000 Pyruvate: 370,000 ATP: 2,000,000 Ca ions: 2,300,000 Fe ions: 7,000,000 Data from: http://bionumbers.hms.harvard.edu http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi David S. Goodsell (Scripps)

12. E. Coli Statistics Protein abundance per cell: ATP Dependent helicase: 104 LacI repressor: 10 to 50 molecules LacZ (galactosidase) : 5000 CheAkinase (chemotaxis): 4,500 CheB (Feedback): 240 CheY (Motor signal): 8,200 Chemoreceptors: 15,000 Glycolysis Phosphofructokinase: 1,550 PyruvateKinase: 11,000 Enolase: 55,800 Phosphoglyceratekinase: 124,000 Krebs Cycle MalateDehydrogenase: 3,390 Citrate Synthase: 1,360 Aconitase: 1630 E coli has approximately 4300 protein coding genes. Source: Protein abundance profiling of the Escherichia coli cytosol. BMC Genomics 2008, 9:102. Ishihama et al.

13. E. Coli Statistics Protein abundance per cell: ATP Dependent helicase: 104 LacI repressor: 10 to 50 molecules LacZ (galactosidase) : 5000 CheAkinase (chemotaxis): 4,500 CheB (Feedback): 240 CheY (Motor signal): 8,200 Chemoreceptors: 15,000 Glycolysis Phosphofructokinase: 1,550 PyruvateKinase: 11,000 Enolase: 55,800 Phosphoglyceratekinase: 124,000 Krebs Cycle MalateDehydrogenase: 3,390 Citrate Synthase: 1,360 Aconitase: 1630 E coli has approximately 4300 protein coding genes. Molecules Numbers in Prokaryotes: Ions Millions Small Molecules 10,000 – 100,000 Metabolic Enzymes 1000 – 10,000s Signaling Proteins 100 – 1000s Transcription Factors 10s to 100s DNA 1 – 10s Source: Protein abundance profiling of the Escherichia coli cytosol. BMC Genomics 2008, 9:102. Ishihama et al.

14. Circular Chromosome in E. coli Most Prokaryotic DNA is circular. Gene are located on both strands of the DNA. Genes on the outside are transcribed clockwise and those on the inside anticlockwise. E. coli’s genome is 4,639,221 base pairs Coding for 4472 genes, of which 4316 are genes that code for proteins.

15. Circular Chromosome in E. coli 88% of the E. coli genome codes for proteins, the rest includes RNA coding, promoter, terminators etc. In contrast, the Human genome: 3,000,000,000 base pairs and about 25,000 genes. Only 2% of the Human genome codes for proteins. The rest is……RNA regulatory network? Human genes are also segmented into Exon and Introns, with alternative splicing, significantly increasing the actual number of protein

16. EcoCyc: http://ecocyc.org/

17. E. coli Gene Structure Stop codon (TAG, TAA, TGA) Start codon Page 134

18. RNA Polymerase Binds to Promoters mRNA Changes in the promoter sequence can change the efficiency of RNA polymerase binding to the DNA. The promoter is therefore a site which can be engineered. http://mgl.scripps.edu/people/goodsell/pdb/pdb40/pdb40_1.html

19. Strong and Weak Promoters The strength of a promote is one of the factors which determines the rate of transcription. Strong Promoter. The recA promoter is a strong promoter. TTGATA -- 16 -- TATAAT TTGACA -- 17 -- TATAAT Most common Promoter (Consensus sequence) It differs from the averaged promoter sequence by one nucleotide and on base pair in the spacer region. Weak Promoter. The araBAD promoter is a weak promoter. CTGACG -- 18 -- TACTGT TTGACA -- 17 -- TATAAT

20. Changes in the terminator sequence can change the efficiency of RNA polymerase 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 site which can be engineered. RNA Polymerase Stops at a Terminator

21. Operon Structure Gene A Gene B Gene C 100% 60% 30% Promoter Terminator

22. Operators – Regulating Expression

23. Gene Regulationlac Operon Metabolic Enzyme (output) Promoter Promoter Operator lacZ codes for β-galactosidase. lacY codes for β-galactosidepermease.

24. Gene Regulationlac Operon Lac repressor Metabolic Enzyme (output) Promoter Promoter Operator

25. Gene Regulationlac Operon

26. LacIRepressor lacI is a tetramer (x4)

27. LacI binding to Promoter

28. Ribosome Binding Sites

29. In summary: Stop Codon Start Codon Promoter RBS Gene Terminator Operators 5’-UTR

30. This course is about networks: TheScience and Engineering of Biological Networks

31. Electronic The world is full of networks WWW Road Social

32. Biological Networks

33. Metabolic Metabolic Networks About 1000-1400 genes that code for metabolic enzymes in E. coli (out of a total of about 4300 genes)

34. Protein Signaling Network Protein-Protein Networks

35. Protein Signaling Network: CellDesigner Kohn MIMS Protein-Protein Networks 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.

36. C Protein-Protein Networks

37. Gene Regulatory Networks: BioTapestry Genetic Networks

38. Gene Regulatory Networks: BioTapestry : Ventral Neural Tube in Vertebrate Embryo Genetic Networks

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

40. Hybrid Network: Cell Cycle Control is Bacteria

41. Cytoscape: Ball and Stick Non-Stoichiometry – or ball and stick networks No stoichiometry, kinetics or mass conservation 2. Stoichiometry – reaction maps ?? – Stuff that people make up, whose knows what they really mean Two Kinds of Representations Stoichiometric

42. Network Classification

43. Systems and Synthetic Biology Top Down Bottom Up Systems Biology Synthetic Biology

44. Top Down “-omics” Top Down and Bottom Up Yeast Protein-Protein Interaction Map

45. Bottom Up ”mechanistic” Top Down “-omics” Top Down and Bottom Up