Chapter 7: Screening and Identification of Recombinant Clones

1. Chapter 7: Screening and Identification of Recombinant Clones • Cloning Procedure • Transformation • Screening and Selection • Identification • Application

2. References • Chapter 15 in : “Essentials of Molecular Biology” • Chapter 4, 6, 8 in “Introduction to Genetic Engineering”

3. I. Cloning Procedure • Isolation and preparation of DNA for cloning • Choose an appropriate vector • Restriction enzyme digestion • Ligate the insert into the vector with compatible ends • Transformation into host cells • Selection and/or screening for transformants

4. insert Overview of Cloning a DNA Fragment into a Plasmid Vector Bacterial Transformation

5. Target DNA for Cloning • Genomic DNA (isolation and purification) • cDNA converted from mRNA (RT) • PCR-amplified DNA • Recombinant DNA-subcloning

6. Agarose Gel Analysis of Digested DNA Fragments Two types of gels used for separating DNA fragments Agarose琼脂糖gels: very porous; ideal for large fragments (0.5-20 kb) Polyacrylamide聚丙烯酰胺gels: small pores; ideal for small fragments (0.1-1 kb)

7. Circular forms of DNA migrate in agarose differently from linear DNAs of the same mass. The plasmids that differ in conformation uncut cut Molecular Biology

8. The DNA fragment migrate differently when at different agarose concentrations Molecular Biology

9. EcoRI EcoRI • GAATTC • CTTAAG • GAATTC • CTTAAG 1 Digestion • AATTC • G • G • CTTAA 2 Annealing of sticky ends Ligase • AATTC • G • G • CTTAA 4 Recombinant DNA • AATTC • G • G • CTTAA 3 Ligation Digestion and Ligation

10. Example

11. Ampr R. E. Digestion R. E. Digestion pUC18 LacZ Host Cell Transformation of cells with the recombinant plasmid Cloning into pUC18 Matching sticky ends anneal退火 Addition of ligase joins nicks and makes a single recombinant plasmind

12. Directional Cloning方向的

13. Bacteria transfer DNA in 3 ways • Transformation Uptake of foreign DNA from surroundings • Transfection • Conjugation(结合) DNA exchanged through cytoplasmic bridge developed between two cells

14. II. Transformation The process of introducing free DNA in to a host cell. (often E. coli strain K12) • CaCl2-heat transformation: Bacteria cells are treated with ice-cold CaCl2 and then exposed to high temperature (42°C) for about 90 seconds. The exact mechanism of action of CaCl2 heat transformation is not known • Electroporation: A strong electric field used to render致使the host cell wall permeable to the free DNA through the use of equipment called electroporators • Conjugation结合: Natural process by which cell-to-cell genetic transfer

15. E. coli not naturally transformable, must perturb感到不安the membrane using chaotropic引起混乱的agents Competent有能力的: able to take up foreign DNA E. coli transformation- inefficient • Small proportion of cells become competent • 1:10,000 DNA molecules taken up • 1:100,000 linear DNA molecules taken up • Plasmids larger than 10 kb taken up poorly

17. CaCl2-heat Transformation

18. Electroporation Good alternative for bacteria that cannot be transformed using chaotropic agents

19. Electroporator Electroporator can generate high frequency pulses to generate transient opening on cells by which the DNA molecules enter the cell

20. III. Screening and Selection • Selection for single DNA fragment cloning • Screening for library to find the one you are interested

21. Selection for single DNA fragment cloning ——Blue white screen The principles of blue white screen Molecular Biology

22. Gene Inactivation

23. Identify colonies with gene of interest (Blue/white screening and selection) After transformation Plate on selective medium to find colonies with cloned DNA E. coli with cloned DNA Selection

24. Molecular Biology

25. Screening for library to find the one you are interested

26. Screening ——Display technology • The introduction of display technology • The principles and the methods of display technology • The characteristic of display technology • The classification of display technology Molecular Biology

27. The introduction of display technology • Display technology is a high-throughput screening techniques that are used to screening the gene of interest and mutation gene. • According to the different display system it can be categorized into phage display technology, yeast display, bacteria surface display, and so on. Molecular Biology

28. The introduction of Flow cytometry Molecular Biology

29. The laser source and beam light system Molecular Biology

30. The conceptual diagram of fluorescence signal Molecular Biology

31. Right Angle Light Detector a Cell Complexity Incident Light Source Forward Light Detector a Cell Surface Area The conceptual diagram of Incident Light Source Molecular Biology

32. The principles and the methods of display technology Interest gene Screening the functional protein from the library Molecular Biology

33. The principles and the methods of display technology Interest gene Screening the higher affinityprotein Molecular Biology

34. The characteristic of the display technology anchoring protein Molecular Biology genotype phenotype

35. The classification of display technology ——Phage display technology 噬菌体编码10个基因p（I，II，III，IV，V，VI，VII，VIII，IX，X ）， 其中III，VI，VII，VIII 和IX 编码衣壳蛋白。在噬菌体展示技术中，常将展示基因与pIII或pVIII 基因相融合。 Molecular Biology

36. 洗脱后再扩增培养 Interest gene Molecular Biology

37. The classification of display technology —— yeast display 酵母是具有细胞壁的单细胞真核微生物。细胞壁分为两层, 内层提供细胞壁的强度, 由β-1 , 3 葡聚糖和β-1 , 6 葡聚糖与少量几丁质组成。外层由甘露糖蛋白组成, 决定了大多数细胞的表面特性。 利用凝集素蛋白(一种甘露糖蛋白)与目的蛋白融合,展示在酵母细胞表面. Interest gene 加入荧光素标记的抗原 流式细胞仪筛选 Molecular Biology

38. The classification of display technology——bacteria surface display Molecular Biology

39. Molecular Biology

40. IV. Identification of Recombinant Molecules • Restriction Digestion and Gel electrophoresis • Nucleic acid hybridization • Immunoblot analysis • DNA sequencing • Or ??(take home message)

41. V. Application • Single DNA fragment cloning • Gene expression • Library construction • cDNA • Genomic

42. 1. Single DNA Fragment Cloning • Direct cloning • Subcloning • PCR cloning

43. Direct cloning 10_21_DNA ligase.jpg

44. Example of Subcloning

45. 2. Gene Expression • Recombinant protein expression • Transgenic research • Gene therapy

46. Recombinant Protein Expression Immunofluorescence red -- alpha tubulin green -- vimentin (cytoskeletal protein) blue -- DNA

47. GFP reporter in adult mice Transgenic mouse harboring a fusion of a muscle precursor-cell specific promoter to lacZ The sea anemone Anemonia sulcataGFP resources promoter from lacZ

48. A transgenic mouse that expresses a defective version of a helicase involved in DNA repair – leads to premature aging 10_39_Transgenic.mice.jpg Shows that accumulation of DNA damage contributes to the aging process

49. Summary • Components for DNA cloning: vector, DNA of interest, restriction enzymes, DNA ligase and competent cells • Transformation: CaCl2-heat, electroporation • Two types of library: genomic and cDNA based on the sources of DNA cloned • Selection and/or screening are the key+