실험 3. Isolation of M13 phage and determination of plaque forming unit

[colony]

I. Introduction

1. Biology to filamentous bacteriophage

1) Genome of wild type bacteriophage M13 (Fig.6-1)
2) Gene product VIII - major structural protein
3) Gene product III - minor coat protein
4) Intergenic region - regulate gene expression‎ and viral DNA synthesis
5) Gene product II - generate unit length viral genome
 

2. Replication of bacteriophage M13

Single stranded circular DNA - RF DNA - (-)RF DNA is transcribed into viral mRNA - nick in the (+)RNA - (+)strand is synthesized by rolling circle replication - (+)strand is cleaved and circularized - progeny (+)strand
 

3. Bacteriophage M13 vectors - M13mp18/19

4. Bacterial host for M13 vectors 

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M13 enter the host cell through sex pili encoded by an F factor. only male bacteria are used to propagate the virus. - TG1, JM109, XL1-Blue, KK2186
 

5. Uses of single strand DNA generated by bacteriophage vectors

1) DNA sequencing by dideoxy chain termination method
2) Generating DNA probes for hybridization that are radiolabeled in only one
     strand
3) Site-directed mutagenesis

II. Materials

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2 x YT medium, per liter (pH 7.0)
         Bacto-tryptone       16g
         Yeast extract          10g
         NaCl                         5g

Minimal(M9) medium : Per liter
         5x M9 salts          200ml
         1M MgSO₄               2ml
         20% glucose         20ml
5x M9 salt: Na₂HPO_4·7H₂O 64g, KH₂PO₄ 15g, NaCl 2.5g, NH₄Cl 5g
The salt solution and MgSO₄ solutions should be prepared separately, sterilized by autoclaving. Glucose should be sterilized by filtration.

Top agar : Tryptone 10g, Yeast extract 5g, NaCl 5g, Agar 7g (per liter)
LB plates
TG-1, XL1-Blue, CJ236, of JM109 : a bacterial strain carrying an F' episome
M13KO7 (helper phage)
Kanamycin 40mg/ml in H₂O
15ml culture tube and sterile toothpick

III. Experimental Procedures

1. Preparation fo plating bacteria

 

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Streak a master culture of a bacterial strain carrying an F' episome (e.g. TG-1) into a minimal (M9) agar plate. Incubate for 24-36 hr at 37℃.
Inoculate 50 ml of LB medium in a 250 ml flask with a single well-isolated colony picked from the minimal agar plate. Agitate the liquid for 6-8 hr at 37℃ in a rotatory shaker until the OD₆₀₀ reaches 0.8
 

2. Picking and growth of M13KO7

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Dispense 2-3 ml of 2x YT medium containing kanamycin (70ug/ml) in a 15 ml culture tube.
Touch the surface of the chosen plaque with the end of a sterile toothpick, and then drop the toothpick into the tube. Incubate for 16-18 hr at 37℃ with moderate agitation (250 cycles/minute).
Transfer the cells to a sterile microfuge tube, and centrifuge at 12,000 rpm for 2 min at 4℃ in a microfuge tube and store at 4℃ .
Measure the titer of the bacteriophage stock by the plaque formation on E. coli strain(e.g. TG-1) that supports the growth of bacteriophage M13.
 

3. Plating bacteriophage M13 and Plaque forming unit determination

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Prepare a series of labeled, sterile tubes containing 5 ml of melted top agar (made up in LB medium). Store the labeled tubes in a water bath at 45℃ .
Prepare 100 fold serial dilutions of the bacteriophage stock in LB medium. Dispense 100 ul of each dilution to be assayed into each of two labeled tubes.
Add 100 ul of plating bacteria to each tube. Mix by vortexing gently.
Pour the mixture onto a labeled plate containing 30-50 ml of hardened LB agar medium, equilibrated to 37℃. Swirl the plate gently to ensure an even distribution of bacteria and top agar.
Allow the top agar to harden for 5 min at RT. Invert the plates and incubate 37℃. Plaques first appear after about 4-5 hr at 37℃ and are best visualized after 8 hr of incubation.

IV. Notes

1. If cells are grown at temperature below 34℃, sex pili of host cells do not form and phages cannot infect.
2. M13 can diffuse considerable distance through top agar. To minimize the possibility of cross contamination it is therefore important to pick plaques that are well separated (-1cm) from their nearest neighbors. Do not pick plaques that have been grown for more than 12 hr at 37℃ or stored for extended periods at 4℃.
3. Stationary phase cells tend to lose F' episomes. The plating culture should therefore be chilled to 0℃ and placed in storage at 4℃ before it reaches saturation. Plating bacteria can be stored in this fashion for periods up to 1 week.
4. A bacteriophage M13 plaque contains between 10⁶ and 10⁸ pfu. Medium from a liquid culture of bacteria infected with bacteriophage M13 contains between 10¹⁰ and 10¹² pfu/ml.
5. During propagation of M13KO7, there is selection for bacteriophage genomes that have lost the p15A origin and the Tn903 transposon. It is therefore important not to pass the bacteriophage serially and to use for superinfection stocks of M13KO7 derived directly from a single plaque.

V. References

1. Hackett  P. B., Fuchs J. A., and Messing J. W. 1987. An introduction to recombinant DNA techniques (2nd edition). The Benjamin/Cummings Publishing Co. Inc.
2. Sambrook J., Fritsch E. F., and Maniatis T. 1989. Molecular cloning (2nd edition). Cold Spring Harbor Lab. Press. 41-54.
3. Lewin B. 1990 Gens VI. Cell Press, Oxford. 336-344.
4. Gibson T. J. 1984. Sudies on the Epstein-Barr virus genome. Cambridge University, England.
5. Short J. M. et al. Nucleic Acids Res. 16 7583.
6. Vieira J., and J. Messing. 1987. Methods Enzymol. 153. 3

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