Week 7, 322

1. Week 7, 322 Monday: remove female Nasonia Tuesday: no class Wednesday: mt DNA Friday: Bacteria, Conjugation

2. Monday, Nasonia • Gently tap the fly pupae onto a folded piece of paper, • Tap adult wasps into the Morgue, • Replace the fly pupae in the same tube, place in incubator of choice, 18, 28 or room temp ~24o C.

3. Mitochondrial DNA • 16, 569 bp, • multiple copies per mt, • 100 - 1000 mt per cell, • 37 genes; • 22 oxidative phosphorylation, • 13 tRNA, • 2 rRNA, • Mitochondrial Control Region.

4. Mitochondrial Control Region • control region, • single promoter on each strand initiates transcription, • ori, • D-loop, • replication loop topography, • hypervariable region, • mutation rate 10x greater than genome.

5. Mitochondrial Control Region • Hair follicle DNA extraction, • PCR, • Sequencing (at Cold Spring Harbor), • Sequence analysis here at WWU. Link Out

7. Bacteria Do Almost Everything Bacteria Predominate • Metabolism; • Phototrophs, • Chemotrophs, • Biochemistry; • ‘fix’ or synthesize a huge range of molecules, • break down almost anything, • adapt to just about anything. • Molecular Biology; • Clone, • Gene therapy, • Eugenics, • Biotechnology, • Etc. • 10,000+ “Species”, • Mycoplasma genetalium • 200 nm • Thiomargarista namibiensis • 750 mm • soil, water, air, symbionts, • have adapted to aquatic and terrestrial extremes, • 100 grams/person, • 1014 bacteria.

8. Bacterial Chromosome • ...a circular molecule of double helical DNA, • 4 - 5 Mb long in most species studied, • 1.6 mm long if brokenand stretched out. • Inside the cell, the circular chromosome is condensed by supercoiling and looping into a densely packed body termed the nucleoid.

9. Extra Chromosomal DNA • Plasmids: circular double stranded DNA molecule that replicates independently, • containing one or more (non-essential) genes, smaller than the bacterial chromosome, • may carries genes for pathogenicity, • may carry genes for adaptation to the environment, including drug resistance genes, • 1000’s of base pairs long.

10. Bacterial Model OrganismEscherichia coli = E. coli • Enteric bacteria: inhabits intestinal tracts, • generally non-pathogenic, • grows in liquid, • grows in air, • E. coli has all the enzymes it needs for amino-acid and nucleotide biosynthesis, • can grow on minimal media (carbon source and inorganic salts), • Divides about every hour on minimal media, • up to 24 generations a day,

11. Growth Equals Cell Division

12. DNA Replication

13. Bacterial mitosis. Why don’t bacteria do meiosis?

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

15. Counting Bacteria 10-3 10-4 10-5 (Serial) Dilution is the Solution

16. Model Model Organism • Ease of cultivation, • Rapid Reproduction, • Small size, • Fecund (large brood size), • Mutants are available, stable and easy to identify? • Literature? • PubMed Listings: Eubacteria: 612,471, Archaebacteria: 9,420

17. Bacteria Phenotypes • colony morphology, • large, small, shiny, dull, round or irregular, • resistance to bactericidal agents, • auxitrophs, • unable to synthesize raw materials from minimal media, • cells unable to break down complex molecules, • essential genes, usually studied as conditional mutants.

18. 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.

19. 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)

20. 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).

21. 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.

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

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

24. F Pilus Attaches to F- Cell

25. F Factor Replicates During Binary Fission

26. 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.

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

28. 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).

29. 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

30. Selective Media • wild-type bacteria grow on minimal media, • media supplemented with selected compounds supports growth of mutant strains, • minimal media + leucine supports leu- cells, • minimal media + leucine + arginine supports leu- arg- • etc. • Selective Media: a media in which only the desired strain will grow, • Selective Marker: a genetic mutation that allows growth in selective medium.

31. Selection ...the process that establishes conditions in which only the desired genotype will grow.

32. 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.

33. 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:

34. 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:

35. Announcement • No class Monday, Tuesday 13th. • - lecture topic will be presented the preceding week. Extra Credit • On another piece of paper, answer the dilution problems on the last page of your handout (2 pts).