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Arabidopsis Experiments:

Arabidopsis Experiments:. Forward Genetic Screen (Ethylene Insensitive Mutants): requires thinking. II. Reverse Genetic Screen / PCR Genotyping (H + - ATPase Mutants): requires scoring F2 and thinking. dominant. recessive. What Next? …experiment I. Thought Experiments….

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Arabidopsis Experiments:

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  1. Arabidopsis Experiments: Forward Genetic Screen (Ethylene Insensitive Mutants): requires thinking. II. Reverse Genetic Screen / PCR Genotyping (H+- ATPase Mutants): requires scoring F2 and thinking.

  2. dominant recessive What Next?…experiment I Thought Experiments… • Backcross to wild-type, • what might the F1 and F2 tell us? • Complementation tests? • Given etr1, etr2, ers1, ers2, ein4, ctr1, ein2, ein3, eil1 and erf1 homozygous plants, and wt plants; devise a plan to describe the genetic nature of the 12 long hypocotyl mutants you found.

  3. L t T homozygote wt L t T heterozygote 5’ 5’ 5’ 3’ 3’ 3’ L t T 5’ 5’ 5’ 3’ 3’ 3’ homozygote mutant What Next?…experiment II

  4. TT TT TT Tt Tt Tt T T t t T t Tt Tt Tt tt tt tt T T T t t t 1 : 2 : 1 1 wt : 2 het 1 wt : 1 het Not Lethal Lethal Gametophyte Lethal Genetic AnalysisF2 Segregation (Friday)…what next? How would you confirm / extend F2 results?

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

  6. 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.

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

  8. 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.

  9. 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.

  10. 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)

  11. 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).

  12. 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.

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

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

  15. F Pilus Attaches to F- Cell

  16. F Factor Replicates During Binary Fission

  17. 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.

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

  19. 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).

  20. 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.

  21. 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

  22. 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:

  23. 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:

  24. What’s Growing?…mate in rich, transfer to… • Plate: minimal + glucose + streptomycin + thiamine: • CSH23 yes / no • CSH50 yes / no • exconjugate yes / no • Plate: MacConkey (rich) + streptomycin plates: • CSH23 yes / no • CSH50 yes / no • exconjugate yes / no CSH23 F’lac+ proA+proB+ D(lacpro)supE spcthi x CSH 50: araD(lacpro)strA thi  F’lac+ proA+proB+ araD(lacpro)strA thi

  25. Extra Credit • On another piece of paper, answer the dilution problems on the last page of your handout (2 pts), due with your abstract on Weds.

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