1 Advanced Gene Technology

2 1 st Lecture: Chapter 4: Creation and evaluation of different gene libraries 2 nd Lecture: Chapter 4 +5: Screening of libraries, specific applications of PCR and analysis of DNA 3 rd Lecture: Chapter 6: General Considerations of gene expression 4 th Lecture: Chapter 6: Gene Expression Systems in Prokaryotes (E. coli, Bacillus, other prokaryotes) 5 th Lecture: Chapter 7: Gene Expression Systems in Eukaryotes (Yeast, insect cells, and mammalian cells) 6 th Lecture: Chapter : Transgenic Animals and Plants 7 th Lecture: Chapter 8: Protein Engineering + HTS screening methods for improved enzymes [Reymond: Chapter 6 (HTS screening and selection of Enzyme-encoded genes) Arnold: Chapter 8 (Evaluating a screen and analysis of mutant libraries) ] 8 th Lecture: Introduction to HTS; screening for novel enzymes + biophysical HTS methods [Hueser: Chapter 2 (HTS for target lead discovery) ; HTS: Chaper 6 (Cellular Assays in HTS) + 9 (Natural products or not?) Reymond: Chapter 8 (Molecular approaches for the screening of novel enzymes) ] 9 th Lecture: HTS: Combinatorial chemistry [Hueser: Chapter 9 (Combinatorial Chemistry and HTS) ] 10 th Lecture: HTS: Immobilization of DNA and enzymes, array technology – part 1 [Reymond: Chapter 12 (Enzyme assays on chips) ] 11 th Lecture: HTS: Immobilization of DNA and enzymes, array technology – part 2 [Reymond: Chapter 12 (Enzyme assays on chips) ] + HTS: Screening automation technology [Hueser: Chapter 3 (Tools and technologies that facilitate automated screening) ] 12 th Lecture: Chapter 20: Human Molecular Genetics 13 th Lecture: Chapter 9: Molecular Diagnostics 14 th Lecture: Chapter 10: Therapeutic Agents 15 th Lecture: Chapter 11: Vaccines Lecture Plan:

3 Books: -> Molecular Biotechnology, by Glick and Pasternak, ASM Press Additional books: -> Gene Expression Systems by J. M. Fernandez & J. P. Hoeffler, Academic Press -> Protein Engineering and Design by P. R. Carey, Academic Press -> Principles of Gene Manipulation by S. B. Primrose et al., Blackwell Science -> From Genes to Genomes by J. W. Dale & M. von Schantz, Wiley -> Prokaryotic Gene Expression by S. Baumberg, Oxford University Press Books for HTS: 1. “HTS in drug discovery” by Hueser, Wiley, “Enzyme assays” by Reymond; Wiley, “High throughput screening”; Methods in Mol. Biol., Humana press, “Directed enzyme evolution: Screening and selection”; Humana press, 2003 Necessary book chapters on HTS under:

4 DNA,RNA, Recombinant DNA Technology

5 Nucleic Acids – DNA and RNA

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7 DNA 1°structure -> sequence Enzymatic chain elongation always 5’-> 3’

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9 The central dogma

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11 What is a gene?

12 OperonExon - Intron

13 The genetic code

14 DNA Polymerase mRNA Ribosome Protein

15 Recombinant DNA Technology

16 Recombinant DNA Technology Clones -> Cells or organisms with identical DNA

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18 Gel Electrophoresis X-Ray structure of a complex of ethidium bromid with DNA.

19 Construction of recombinant DNA molecule

20 Ligation conditions Temperature: 4º-10ºC -> takes long time 16ºC -> good temperature, but maybe inconvenient RT (room temp) -> much faster, compromise Concentration of DNA : high -> intermolecular (between different molecules) ligation fevered low -> intramolecular (within one molecule) ligation fevered Optimal vector-insert ratio: from 1:3 to 3:1 (molar ratio -> vector: insert) depending on size e.g.: vector: 5kb + insert: 500 bp -> molar ratio of 1:1 -> 500ng vector + 50 ng insert vector: 6kb + insert: 50kb -> 1:1 -> 500ng vector + 5ug insert -> W V /S V :W I /S I

21

22 Plasmid Cloning Vectors

23 Insertional inactivation Gene in cloning site: LacZ -> pUC18 (lacZ complements the host defect in lacZ) -> pUC18 into host organism -> active lacZ (β-galactosidase) from plasmid-> cleavage of X-gal (blue colonies) -> gene cloned into polylinker -> lacZ gene disrupted -> no cleavage of X-gal (white colonies)

24 Insertional inactivation Gene in cloning site: Resistance marker -> pBR322 (cloning sites within antibiotica resistence marker) -> plasmid into host -> resistance against 2 antibiotica -> gene cloned within one resistance marker -> gene for antibiotica resistance marker disrupted -> sensitive against one antibioticum

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26 Transformation and Selection

27 Horizontal gene transfer - Transformation -> uptake of naked DNA (chemical transformation, electroporation) - Conjugation -> DNA transfer by cell – cell contact - Transduction -> DNA transfer by bacteriopage infection Other methods of Gene transfer -> used with fungi, animal and plant cells: - Microinjection - protoplasts

28 Electron micrograph of bacteriophage λ. Electron micrograph of the filamentous bacteriophage M13. Bacteriophages Bacteriophage T2 injecting its DNA into an E. coli

29 Life Cycle of Bacteriophage λ

30 Regulation of Life Cycle of Bacteriophage λ cI repressor  prevents lytic cycle

31 This autoregulation of cI synthesis keeps the cell in a stable state of lysogeny. If this is the case, how do such cells ever leave this state and enter a productive, lytic replication cycle? Physiological stress and particularly ultraviolet irradiation of cells results in the induction of a host cell protein, RecA. This protein, whose normal function is to induce the expression of cellular genes which permit the cell to adapt to and survive in altered environmental conditions, cleaves the cI repressor protein. In itself, this would not be sufficient to prevent the cell re-entering the lysogenic state. However, when repressor protein is not bound to OR, cro is transcribed from PR. This protein also binds to OR, but unlike cI, which preferentially binds to the right-hand end of OR, the cro protein binds preferentially to the left-hand end of OR, preventing the transcription of cI and enhancing its own transcription in a positive feedback loop. Thus, the phage is locked into a lytic cycle and cannot return to the lysogenic state. Regulation of Life Cycle of Bacteriophage λ

32 Replication of bacteriophage upon infection of a cell

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34 Plaques – lyst cells apon infection with bacteriophages

35 Cloning of foreign DNA in λ phages.

36 What is a gene library ?

37 Creation of Libraries

38 Sizes of Some DNA Molecules. Page 92

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40 Phage library In vitro packaging in Phage λ heads: -> two proteins Nu1 and A can recognize the COS site, directing the insertion of the DNA between them into an empty head -> Capacity: 10 – 20 kbps -> higher transformation efficiency than plasmid library

41 Vector system: Cosmid = Cos - Plasmid In vitro packaging in Phage λ heads: -> two proteins Nu1 and A can recognize the COS site, directing the insertion of the DNA between them into an empty head Advantage: -> High transformation efficiency. -> The cosmid vector can carry up to 45 kb whereas plasmid and phage vectors are limited to 25 kb.

42 Vector system: BAC (Bacterial artificial chromosome)

43 Vector system: YAC (Yeast artificial chromosome) -> TEL: The telomere which is located at each chromosome end, protects the linear DNA from degradation by nucleases. -> CEN: The centromere which is the attachment site for mitotic spindle fibers, "pulls" one copy of each duplicated chromosome into each new daughter cell. -> ORI: Replication origin sequences (ARS – autonomous replicating sequence in Yeast) which are specific DNA sequences that allow the DNA replication machinery to function. Two copies of the Washington University Human Genome YAC Library. Each of the stacks is approximately 12 microtiter plates. Each plate has 96 wells, each with different yeast clones.

44 Fosmids are similar to cosmids but are based on the bacterial F-plasmid cosmidsbacterialF-plasmid

45 Library: Different type of Insert

46 Fragmentation of genomic DNA

47 cDNA synthesis

48 Evaluation of library

49 Evaluation of library

50 Ordered library Microarrays

51 Ordered library “Chromosome Walking” -> also used in “Human Genome Project”

52 Different ways to clone a gene