Microbial genetics and genetic engineering

1. Microbial genetics Part 3

2. MUTATION

3. Genetic Engineering Recombinant DNA technology, gene cloning

4. Genetic Engineering Genetic engineering involves changing the genetic material in an organism to alter its traits or products A recombinant DNA molecule contains DNA fragments spliced together from 2 or more organisms

5. Outline Stages of cloning experiment Elements: Vectors Restriction enzymes Mechanism in joining the fragments Selection or detection of successful cloning Gene library

6. Stages of Cloning 1. Joining DNA segment 2. Providing milieu that allows propagation Clones Vector

7. Recombinant Bacteria Remove bacterial DNA (plasmid). Cut the Bacterial DNA with “ restriction enzymes ”. Cut the DNA from another organism with “ restriction enzymes”. Combine the cut pieces of DNA together with another enzyme and insert them into bacteria. Reproduce the recombinant bacteria. The foreign genes will be expressed in the bacteria.

8. Vectors : properties It must be able to replicate There must be some way to introduce vector DNA into the cell There must be some way of detecting its presence, preferably by plating techniques Most common vectors are: Plasmid, phage λ and viruses

9. Restriction Enzymes Discovered in an experiment where bacteriophage lost its plaque formation in E.coli Enzymes that recognize a specific base sequence in a DNA molecule It makes two cuts, one in each strand, generating 3’-OH and 5’-P termini Types of termini produced: Flush or blunt end Cohesive or sticky end

10. Examples of Restriction Enzymes

11. Other features of Restriction Enzymes The number of cuts made in the DNA from specific organism is limited A particular restriction enzyme generates a unique family fragments from a DNA molecule

12. • Bst EII pUC19 pUC19 • Hin dIII  • Hin dIII • Bst EII

13. Restriction Mapping Restriction maps show the relative location of a selection of restriction sites along linear or circular DNA. HindIII BamHI PstII PstII BamHI HindIII EcoRI

14. Simple example Digests 1:  EcoRI 2:  HindIII 3:  EcoRI  +  HindIII   Resultant Fragments: approximate sizes 1: 3 kb, 5 kb 2: 6 kb, 2 kb 3: 2 kb, 1 kb, 5 kb,

15. Restriction Mapping BglII BamHI PstI BglII +BamHI BglII +PstI BamHI +PstI 4.2 5.2 3.6 3.5 3.3 2.6 1.7 1.7 1.4 1.4 1.2 1.2 1.0 1.0 1.2 0.7 0.9 0.5 0.3 0.3 0.3 BglII BamHI PstI BglII PstI 0.3 0.7 2.6 0.9 0.5 1.2

16. Restriction problems A )  11, 6, 5 B )  14,8 C )  16,6 A  x  B ) 8, 6, 5, 3 A  x  C ) 11, 5, 5,  1 B  x  C ) 8, 8, 6

17. Joining fragments Cohesive end Blunt end Increasing the DNA concentration and addition of ligase Addition of homopolymers Using of linkers

18. Increasing DNA concentration

19. Homopolymers

20. Linkers

21. Selection/detection Antibiotic resistance marker Insertional inactivation Electrophoresis hybridization

22. Gene libraries collection of all of the vector molecules, each carrying a piece of the chromosomal DNA of the organism

23. Applications