7, Week 322
Monday: remove female Nasonia Tuesday: no class Wednesday: mt DNA Friday: Bacteria, Conjugation
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.
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.
Mitochondrial Control Region
controlregion,
singlepromoteroneachstrand initiatestranscription, ori,
Dloop,
replicationlooptopography, mutationrate10xgreaterthan genome.
hypervariableregion,
Mitochondrial Control Region HairfollicleDNAextraction, PCR, Sequencing(atColdSpring Harbor), Sequenceanalysishereat WWU.
Link Out
Bacteria Predominate
10,000+ Species,
Mycoplasma genetalium
200 nm
Bacteria Do Almost Everything
Metabolism;
Phototrophs, Chemotrophs,
Biochemistry;
fix or synthesize a huge range of molecules, break down almost anything, adapt to just about anything.
Thiomargarista namibiensis
750 m
soil, water, air, symbionts, have adapted to aquatic and terrestrial extremes,
Molecular Biology;
Clone, Gene therapy, Eugenics, Biotechnology, Etc.
100 grams/person,
1014 bacteria.
Bacterial Chromosome
...a circular molecule of double helical DNA,
4 - 5 Mb long in most species studied, 1.6 mm long if broken and stretched out.
Inside the cell, the circular chromosome is condensed by supercoiling and looping into a densely packed body termed the nucleoid.
Extra Chromosomal DNA
Plasmids: circular double stranded DNA molecule that replicates independently,
containing one or more (nonessential) genes, smaller than the bacterial chromosome, may carries genes for pathogenicity, may carry genes for adaptation to the environment, including drug resistance genes, 1000s of base pairs long.
Bacterial Model Organism
Escherichia 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,
Growth Equals Cell Division
DNA Replication
Binary Fission
Bacterial mitosis. Why dont bacteria do meiosis?
The (Awesome) Power of Bacterial Genetics
... is the potential for studying rare events.
Liquid Cultures, 109cells/microliter,
Colonies on Agar, 107+ cells/colony
Counting Bacteria
10-3
10-4
10-5
(Serial) Dilution is the Solution
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
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.
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.
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- ...cant synthesize histidine (his+ = wt) leu- ...cant synthesize leucine (leu+ = wt) arg- ...cant synthesize arginine (his+ = wt) bio- ...cant synthesize biotin (bio+ = wt)
Bacterial Nomenclature
genes not specifically referred to are considered wildtype,
Strain A: met bio (require methionine and biotin) Strain B: thr leu thi
bacteriacide resistance is a gain of function,
Strain C: strA (can grow in the presence of strptomycin).
Conjugation
...temporary fusion two of 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.
F
+
F Pilus
F
-
a filamentlike projection from the surface of a bacterium.
F Factor
a plasmid whose presence confers F+, or donor ability.
F Pilus Attaches to F Cell
-
F Factor Replicates During Binary Fission
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.
Hfr Cells
...F factor integration site, ...host (bacteria chromosome) integration site. Bacterial Chromosome
F factor
Inserted F plasmid
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,
Fcells behave like an F+ cells,
merizygote: partially diploid for genes copied on the Fplasmid,
Fplasmids can be easily constructed using molecular biology techniques (i.e.vectors).
Strain CSH23
F genotype Flac+ proA+ proB+ x
Chromosome Genotype (lacpro)supE spc thi
CSH 50:
ara (lacpro)strA thi Conjugation
Recombinant Strain:
Flac+ proA+ proB+
ara (lacpro)strA thi
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.
Selection
...the process that establishes conditions in which only the desired genotype will grow.
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.
Procedure I:
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...