This Week, Nov 10 th Tuesday: no class Thursday: Bacteria, Conjugation Next Week Monday, 17 th : No class Tuesday, 18 th : Collect/Analyze Conjugation.

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

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. 2 = 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. 2 = 0.26, p = 0.8728

Genetic Selection

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

Selective Media: what might this be?

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.

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

(Serial) Dilution is the Solution

10-3 10-510-4

Extra Credit: On another piece of paper, answer the dilution problems on the last page of your handout (2 pts), due Thursday, 13th.

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- ...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)

Bacterial Nomenclature

• genes not specifically referred to are considered wild-type,

– 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 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 donate genetic material.

… F- cells receive genetic material,

…there is no reciprocal transfer.

F- Cells(F for Fertility)

F Pilus

…a filamentlike projection from the surface of a bacterium.

F+

F-

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

Bacterial Chromosome

Inserted F plasmid

...F factor integration site,

...host (bacteria chromosome) integration site.

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).

Transfer of lac+pro+ from a F' to an F- strain.

• Strain Sex Genotype

• CSH23 F’ lac+ proA+ proB+ (lacpro) supE spc thi

• CSH50 F- ara (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.

•

Strain F’ genotype Chromosome Genotype

CSH23 F’lac+ proA+ proB+ (lacpro)supE spc thi

x

CSH 50: ara (lacpro)strA thi

Conjugation

Recombinant Strain: F’lac+ proA+ proB+ ara (lacpro)strA thi

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 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 shaking incubator 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 II:• 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.

Extra Credit

• On another piece of paper, answer the dilution problems on the last page of your handout (2 pts), due Thursday, 13th.

Announcement

No class Monday, Tuesday 17th.