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Bacterial genetics

Bacterial genetics. by E. Börje Lindström. This learning object has been funded by the European Commissions FP6 BioMinE project. Definitions. Gene:. a DNA sequence that determines . the aa-sequence in a peptide/protein rRNA tRNA operator (O) promoter (P) . Allele:.

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Bacterial genetics

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  1. Bacterial genetics by E. Börje Lindström This learning object has been funded by the European Commissions FP6 BioMinE project

  2. Definitions • Gene: a DNA sequence that determines • the aa-sequence in a peptide/protein • rRNA • tRNA • operator (O) • promoter (P) • Allele: - a different form of a gene (wt, mutant) • Genotype: - the total amount of genes in an organism • Phenotype: - those properties that are expressed

  3. Definitions, cont. • Mutation: • a permanent change in the genotype • spontaneous (frequency 10-10—10-5) • induced (frequency >10-5) • Mutant: - an organism that has a changed genotype • Two types of mutants: • Gain of a property • (possible to select for) • Loss of a property • (not selectable)

  4. How to find the mutant? • Those who are selectable: • e.g. Antibiotic sensitive  resistant • streak out the culture on a plate containing the antibiotic. • Those who are not selectable: - e.g. Prototroph (wt)  auxotroph (mutant) • Enrichment with the e.g. penicillin-method • Screening with ’replica plating’

  5. Penicillin-method Inoculum - Only prototrophs can grow  are killed MM-medium + pen - Change the medium • All bacteria will grow • both prototrophs and auxotrophs • the auxotrophs are enriched Complex medium without penicillin

  6. Replica plating

  7. Replica plating, cont.

  8. Nomenclature Model system: tryptophan • Genotype: - trp- (trp+); (A, B, etc.) • Phenotype: - Trp+; Trp- • Amino acid: try

  9. Transfer of DNA between bacteria Donor Recipient Three processes: • Transformation: - f ree donor-DNA  recipient 2) Transduction: - donor-DNA within a virus (Phage)  recipient 3) Conjugation: - cell-to-cell contact between donor and recipient

  10. Transformation

  11. Transformation, cont. The Donor The Recipient - Cultivate, make competent cells - lyse and cut the DNA into pieces Free DNA (10 genes/ fragment) ds DNA - Uptake of DNA (ds or ss) - homologue integration - transformed cell

  12. Transduction Two types: • General transduction • Model system: P1- E. coli • P1 a virulent phage • Specific transduction - Model system: phage l – E. coli

  13. General Transduction

  14. Specifik transduction

  15. Specifik transduction, cont.

  16. Conjugation Three different bacteria: F-plasmid Integrated pili Free Hfr (high frequency of recombination) F- F+ Recipient (female) Donors (males)

  17. Integration of the F-plasmid oriT IS-element

  18. Cross no. 1 • F+ x F- F-  F+ (all) • The F-plasmid is transferred: • a ss-DNA is transferred • new DNA synthesis occur in both cells • the complete F-plasmid is transferred • rapid spread of the F-plasmid

  19. Cross no. 1, cont.

  20. Cross no. 1, cont.

  21. Cross no. 2 • Hfr x F- F-  F- (all) Course of events: 1) Cell-to-cell contact - pili promote a conjugation bridge (Week) 2) ss-brake at oriT in Hfr 3) One of the Hfr-strands is transferred – ori T first • new synthesis in both cells • constant transfer speed (takes 100 min for E. coli) 4) Pairing of homologue DNA-segments 5) Integration and exchange of genetic material 6) Brake down of linear extra-chromosomal DNA 7) A conjugant/ recombinant has been constructed

  22. Cross no. 2, cont.

  23. A- B- A+ B+ C+ Ori T C- Cross no. 2, cont. 40) - Partial diploid 50) A- B- C+ Ori T - Integration and exchange A+ B+ C-

  24. Cross no. 2, cont. Q: Why is streptomycin added to the plate?

  25. Summary • Obtaining a recombinant/conjugant require: • genes must have been transferred from a donor to a recipient • exchange of genes must occur

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