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This Week, Nov 10 th

This Week, Nov 10 th. Tuesday: no class Thursday: Bacteria, Conjugation Next Week Monday, 17 th : No class Tuesday, 18 th : Collect/Analyze Conjugation data Thurday 20 th : Start Hfr Experiment. 19. 20.

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This Week, Nov 10 th

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  1. This Week, Nov 10th Tuesday: no class Thursday: Bacteria, Conjugation Next Week Monday, 17th: No class Tuesday, 18th: Collect/Analyze Conjugation data Thurday 20th: Start Hfr Experiment

  2. 19 20 1. There is no difference between the expected 1:2:1 segregation and the observed values, except as can be attributed to chance. c2 = 18.538, p = <0.0001 2. There is no difference between the expected 1:1 segregation and the observed values, except as can be attributed to chance. c2 = 0.26, p = 0.8728

  3. Genetic Selection ...the process that establishes conditions in which only the desired genotype will grow. Selective Media: what might this be?

  4. Genetic Screen • A system that allows the identification of rare mutations in large scale searches, • unlike a selection, undesired genotypes are present, the screen provides a way of “screening” them out.

  5. Liquid Cultures, • 109cells/microliter, • Colonies on Agar, • 107+ cells/colony The (Awesome) Power of Bacterial Genetics ... is the potential for studying rare events.

  6. Counting Bacteria 10-3 10-4 10-5 (Serial) Dilution is the Solution Extra Credit: On another piece of paper, answer the dilution problems on the last page of your handout (2 pts), due Thursday, 13th.

  7. Bacteria Phenotypes • colony “morphology”, • large, small, shiny, dull, round or irregular, • resistance to bactericidal agents, • vital dyes, • auxitrophs, • unable to synthesize or use raw materials from the growth media.

  8. Prototroph …a cell that is capable of growing on a defined, minimal media, • can synthesize all essential organic compounds, • usually considered the ‘wild-type’ strain. Auxotrophs • …a cell that requires a substance for growth that can be synthesized by a wild-type cell, • his- ...can’t synthesize histidine (his+ = wt) • leu- ...can’t synthesize leucine (leu+ = wt) • arg- ...can’t synthesize arginine (his+ = wt) • bio- ...can’t synthesize biotin (bio+ = wt)

  9. Bacterial Nomenclature • genes not specifically referred to are considered wild-type, • Strain A:met bio (require methionine and biotin) • Strain B:thr leuthi • bacteriacide resistance is a gain of function, • Strain C:strA (can grow in the presence of strptomycin).

  10. Conjugation ...temporary fusion of two single-celled organisms for the transfer of genetic material, …the transfer of genetic material is unidirectional. F+ Cells(F for Fertility) F- Cells(F for Fertility) … F+ cells donate genetic material. … F- cells receive genetic material, …there is no reciprocal transfer.

  11. F+ F- F Pilus …a filamentlike projection from the surface of a bacterium.

  12. F Factor …a plasmid whose presence confers F+, or donor ability.

  13. F Pilus Attaches to F- Cell

  14. F Factor Replicates During Binary Fission

  15. Properties of the F Factor • Can replicate its own DNA, • Carries genes required for the synthesis of pili, • F+ and F- cells can conjugate, • the F factor is copied to the F- cell, resulting in two F+ cells, • F+ cells do not conjugate with F+ cells, • F Factor sometimes integrates into the bacterial chromosome creating Hfr cells.

  16. ...F factor integration site, ...host (bacteria chromosome) integration site. Hfr Cells F factor Bacterial Chromosome Inserted F plasmid

  17. F’Cells • an F factor from an Hfr cell excises out of the bacterial genome and returns to plasmid form, • often carries one or more bacterial genes along, • F’cells behave like an F+ cells, • merizygote: partially diploid for genes copied on the F’plasmid, • F’plasmids can be easily constructed using molecular biology techniques (i.e.vectors).

  18. Transfer of lac+pro+ from a F' to an F- strain. • Strain Sex Genotype • CSH23 F’lac+ proA+ proB+D(lacpro) supE spc thi • CSH50 F- ara D(lacpro)strA thi strA: confers resistance to streptomycin spc: confers resistance to spectinomycin • indicates a deletion of the genes in parentheses lac: cannot utilize lactose as a carbon source pro: indicates a requirement for proline thi; indicates a requirement for thiamine supE: suppresses nonsense mutations ara: cannot utilize arabinose as a carbon source.

  19. Strain F’ genotype Chromosome Genotype CSH23 F’lac+ proA+proB+ D(lacpro)supE spcthi x CSH 50: araD(lacpro)strA thi Conjugation Recombinant Strain: F’lac+ proA+proB+ araD(lacpro)strA thi

  20. Day 0: Overnight cultures of the CSH23 and CSH50 will be set up in L broth (a rich medium). Day 1: These cultures will be diluted and grown at 37o until the donor culture is 2-3 X 108 cell/ml. What is the quickest way to quickly determine #cells per ml? (This will be done for you.) Prepare a mating mixture by mixing 1.0 ml of each culture together in a small flask. Rotate at 30 rpms in a 37o shakingincubator for 60 minutes. At the end of the incubation… Do serial dilutions: Fill 6 tubes with 4.5 ml of sterile saline. Transfer 0.5 ml of the undiluted mating culeture to one of the tubes. This is a 10-1 dilution. Next make serial dilutions of 10-2, 10-3, 10-4, 10-5 & 10-6. Always change pipets and mix well between dilutions. Procedure I:

  21. Plate: 0.1 ml of a 10-2, 10-3 and 10-4 dilution onto minimal + glucose + streptomycin + thiamine. Plate: 0.1 ml of a 10-5 and 10-6 dilution onto a MacConkey + streptomycin plates. [A MacConkey plate is considered a rich media. It has lactose as well as other carbon sources. The phenol red dye is present to differentiate lac+ colonies (red) from lac- colonies (white).] Controls: Plate: 0.1 ml of a 10-1 dilution of donor (CSH23) cells on minimal + glucose + strep + thiamine plates. Repeat for the recipient (CSH50) cells. Plate: 0.1 ml of a 10-5 dilution of the recipient on a MacConkey + strep plate. Plate: 0.1 ml of a 10-1 dilution of donor on a MacConkey + strep plate. Place all plates at 37o overnight. Day 2: Remove the plates from the incubator the next day and count the number of white-clear colonies on the MacConkey plates (optional but easier). Store plates at 4oC. NOTE: MacConkey color reactions fade after several days or rapidly in the cold, so plates need to be scored soon after incubation. Procedure II:

  22. Announcement No class Monday, Tuesday 17th. Extra Credit • On another piece of paper, answer the dilution problems on the last page of your handout (2 pts), due Thursday, 13th.

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