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Budding Technologies and Budding Yeast

Budding Technologies and Budding Yeast. 2012 HHMI Summer Workshop for High School Science Teachers. The Genomics of S.cerevisiae. GOALS. Introduction to the Genomics of Yeast Sequencing Technologies and how they are evolving Introduction to Systems Biology and modern Yeast Genetics.

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Budding Technologies and Budding Yeast

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  1. Budding Technologies and Budding Yeast 2012 HHMI Summer Workshop for High School Science Teachers

  2. The Genomics of S.cerevisiae

  3. GOALS • Introduction to the Genomics of Yeast • Sequencing Technologies and how they are evolving • Introduction to Systems Biology and modern Yeast Genetics

  4. Genetics and Genomics • GENETICS is the science of genes, heredity and variation. • Genetic studies typically focus on a single gene. • Experiments typically involve mutation of the model organism, then looking to figure out what went wrong. • GENOMICS is a discipline of systems biology that focuses on the genome. • Genomic studies typically study all genes at once

  5. Basic Yeast Statistics • 16 chromosomes

  6. Genomic Organization & Nomenclature • 16 Chromosomes. • Range from 230kbp – 1.5Mbp

  7. Basic Yeast Statistics • 16 chromosomes • 13.1 Mbp of sequence E.coli: 4.6 Mbp Yeast: 13.1 Mbp Drosophila: 122 Mbp Human: 3.3 Gbp Zebrafish: 1.2 Gbp

  8. Basic Yeast Statistics • 16 chromosomes • 13.1 Mbp of sequence • 6,183 open reading frames • 73% of the genome codes for genes E.coli: 4,377 Yeast: 6,183 Drosophila: 17,000 Human: 23,000 Zebrafish: 15,800

  9. Basic Yeast Statistics • 16 chromosomes • 13.1 Mbp of sequence • 6,183 open reading frames • 73% of the genome codes for genes • Genes are named by position. Crick Strand Left arm Y A L 014 C Chromosome I 14th gene from the centromere

  10. Where to learn more: • Saccharomyces Genome Database

  11. Where to learn more: Browser • Saccharomyces Genome Database

  12. Yeast as a Model System Yeast share most basic systems with human. • Polymerases • Nucleosomes • Translation • Splicing • Stress response • DNA damage response • Cell Cycle • Mitotic mechanisms • Meiosis

  13. More about Yeast • About75% of yeast genes have something known about them.

  14. More about Yeast • About75% of yeast genes have known functions. • Many genes serve to regulate other genes.

  15. More about Yeast • About75% of yeast genes have known functions. • Many genes serve to regulate other genes. • About 1/3 of proteins are in the nucleus.

  16. GOALS • Introduction to the Genomics of Yeast • Sequencing Technologies and how they are evolving • Introduction to Systems Biology and modern Yeast Genetics

  17. Sequencing the First Eukaryote • 600 Scientists • >100 labs • World wide effort

  18. Sanger Sequencing

  19. Sanger Sequencing

  20. So… How do you sequence a Genome? • Walking

  21. So… How do you sequence a Genome? • Walking

  22. So… How do you sequence a Genome? • Walking • Types of vectors

  23. So… How do you sequence a Genome? • Walking • Shotgunning Completely sequence Randomly fragment Reassemble ~1-2kb

  24. So… How do you sequence a Genome? • Walking • Shotgunning • Mixed Approach • Prescaffolding markers Large vectors

  25. So… How do you sequence a Genome? • Walking • Shotgunning • Mixed Approach • Prescaffolding • Shotgunning the fragments markers Large vectors Small plasmids

  26. Yeast to Human….

  27. A new revolution • 454 • Solexa • ABI

  28. How NGS works • Fundamentally different from Sanger • Detect each base individually, then extend • Watch as polymerase moves along the chain • Each molecule is read multiple times

  29. How NGS works • Illumina Sequencing uses “Sequencing by Synthesis • Adaptors added to DNA to make them bind the flowcell. • In situ, the DNA is amplified into a cluster

  30. How NGS works • Primer then binds to the sequence. • Bases are flowed over the cluster and nucleotides are read.

  31. How NGS works • Primer then binds to the sequence. • Bases are flowed over the cluster and nucleotides are read. • Billions of reads are happening at once.

  32. A new revolution • Sequencing costs are plummeting.

  33. A new revolution • Sequencing costs are plummeting. • Cut in half every year.

  34. A new revolution • Sequencing costs are plummeting. • Cut in half every year. • Yields are sky rocketing.

  35. Applications Re-Sequencing De Novo Sequencing gDNA SNP Discovery Transcript Discovery mRNA Expression Analysis miRNA Analysis miRNA Allelic Expression ChIP-Seq Nuclear run-on IP Copy Number Variation … and more

  36. Applications: Genetics Mutation in alk in 224A/+ D>N homozygous R>H

  37. GOALS • Introduction to the Genomics of Yeast • Sequencing Technologies and how they are evolving • Introduction to Systems Biology and modern Yeast Genetics

  38. Systems Biology • Most molecular biology has been carried out with a reductionist point of view • Look at one gene or one protein or a class of genes • Systems Biology attempts to look at organisms holistically • “OMICS” (genomics, proteomics, metabolomics, transcriptomics, etc.)

  39. Systems Biology: Beginnings • First whole genome experiments were done with microarrays. • Surface of the microarray is spotted with DNA reflecting every gene in the genome • Total RNA is hybridized to the surface • Amount of material can be measured by intensity

  40. Forward Genetics v Reverse Genetics • Forward genetics is the classical method for doing screens. • 1) Find a phenotype. • 2) Find out why it happens. • Reverse genetics mutates a gene, then sees what it does. • This defined genetic alteration makes it amenable to systems biology approaches.

  41. Functional Screen: Two-Hybrid • Screen genome wide for protein interaction partners. • A “prey” library requires every protein to be fused to a transcription activation domain. • Screen with a bait protein that binds to the DNA.

  42. Functional Screen: Two-Hybrid • Screen genome wide for protein interaction partners. • A “prey” library requires every protein to be fused to a transcription activation domain. • Screen with a bait protein that binds to the DNA. • Create large networks.

  43. The Modern Yeast Toolkit • Two-Hybrid • Knockout library • GFP Fusion library • Overexpression library • High Copy • Low Copy • GST fusion library

  44. -factor HU Control Screening GFP Libraries GFP Library STRESS MMS HU a-factor Cntl Protein: RNR4 FIX and STAIN IMAGE Quantify changes in intensityand location Data from Samson Lab

  45. Knockout Library and “BARseq” • Knock out strains have unique molecular barcodes that act as finger prints. • By pooling all the strains together, frequency of each strain can be determined by the frequency of the barcode in NGS experiments

  46. Knockout Library and “BARseq” ALL STRAINS • Experiments can be done by looking at the variations in frequency of the pool after changing the environment of the library. RICH MEDIA MINIMAL MINIMAL + AAs SEQUENCE AND LOOK FOR CHANGES IN FREQUENCY

  47. The Future – Synthetic Biology • Key limitations of current toolset • Have to create each strain separately. • Finite number of mutations being created.

  48. The Future – Synthetic Biology • Assembly of chromosomes in vitro. • Can add any mutation anywhere by replacing a segment and reintroducing. • Can create designer chromosomes with complex and unusual traits • Do not require “carrier markers” Craig Venter, 2010

  49. The End • Introduction to the Genomics of Yeast • Sequencing Technologies and how they are evolving • Introduction to Systems Biology and modern Yeast Genetics

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