Advanced Gene Technology

1. Advanced Gene Technology

2. Recombinant DNA Technology

3. Recombinant DNA Technology Clones -> Cells or organisms with identical DNA

4. Restrictionendonucleases Type II 5’-> 3’ 3’ <- 5’

7. Gel Electrophoresis

8. Gel Electrophoresis

9. X-Ray structure of a complex of ethidium bromid with DNA. Page 1125

10. Construction of a restriction map. Page 104

11. Restriction map for the 5243-bp circular DNA of SV40. Page 104

13. Construction of a recombinant DNA molecule through the use of synthetic oligonucleotide adaptors Page 109

14. 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 -> WV/SV:WI/SI

16. Plasmid Cloning Vectors

17. Plasmid Cloning Vectors

18. 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)

19. 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

21. Transformation and Selection

22. 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

23. Electron micrograph of bacteriophage λ. Bacteriophages Page 107 Electron micrograph of the filamentous bacteriophage M13.

24. Bacteriophage T2 injecting its DNA into an E. coli Page 84

25. Life Cycle of Bacteriophage

28. A simplified genetic map of lambda: • For regulation of the growth cycle of the phage, the structural genes encoding the head and tail components of the virus capsid can be ignored. The pertinent components involved in genetic control are: • PL is the promoter responsible for transcription of the left-hand side of the lambda genome, including N and cIII. • OL is a short non-coding region of the phage genome (approximately 50bp) which lies between the cI and N genes next to PL. • PR is the promoter responsible for transcription of the right-hand side of the lambda genome, including cro, cII and the genes encoding the structural proteins. • OR is a short non-coding region of the phage genome (approximately 50bp) which lies between the cI and cro genes next to PR. • cI is transcribed from its own promoter and encodes a repressor protein of 236 amino acids which binds to OR, preventing transcription of cro but allowing transcription of cI, and to OL, preventing transcription of N and the other genes in the left-hand end of the genome. • cII and cIII encode activator proteins which bind to the genome, enhancing the transcription of the cI gene. • cro encodes a 66 amino acid protein which binds to OR, blocking binding of the repressor to this site. • N encodes an antiterminator protein which acts as an alternative rho factor for host cell RNA polymerase, modifying its activity and permitting extensive transcription from PL and PR. • Q is an antiterminator similar to N, but only permits extended transcription from PR.

29. In a newly infected cell, N and cro are transcribed from PL and PR respectively:

30. 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.

31. Replication of bacteriophage upon infection of a cell Page 107

35. Cloning of foreign DNA in λ phages. Page 110

36. What is a gene library ?

37. Creation of Libraries

38. Sizes of Some DNA Molecules. Page 92

40. Cosmid = Cos - Plasmid

44. Fragmentation of genomic DNA

46. cDNA synthesis

47. DNA Library Clones -> genetically identical

48. Genomic phage library

49. Evaluation of library

50. Evaluation of library