실험 4. Infection, growth, preparation of lambda phage

[colony]

I. Introduction

1. Bacteriophage lambda life cycle

1) RNA replication
2) Assembly
3) Lysogeny
4) Genome structure
 

2. Bacteriophage lambda vector - λgt10, λgt11, λgt18, λgt19, λWES, λEMBL
 

3. Bacterial host for lambda vector - genotypes, phenotype and uses.X
 

4. Long term storage of bacteriophage lambda stocks

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Usual storage : Stable for several years when stored at 4℃ in SM containing a small amount of freshly distilled chloroform(0.3%)
storage of master stocks : Add DMSO-final concentration of 7%(v/v), stored at -70℃
Recover the bacteriophage from storage stocks
 

5. Large-scale preparation of lambda phage

1) Infection at low multiplicity
2) Infection at high muliplicity
 

6. Alternative methods for purification of bacteriophage lambda

1) pelleting of bacteriophage particle
2) Glycerol step gradient
3) Eqilibrium centrifugation in cesium chloride

II. Materials

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Lambda phage
NZCYM medium : per liter
         NZ amine                      10g
         NaCl                               5g
         Bacto-yeast extract        5g
         Casamino acids              1g
         MgSO₄ 7H₂O                  2g
         Adjust the pH to 7.0 with NaOH, sterilize by autoclaving
LB medium
Suspension medium (SM) : per liter
         NaCl                      5.8g
         MgSO₄ ? 7H₂O            2.0g
         1M Tris Cl (pH 7.5)       50ml
         2% Gelatin solution        5ml
         Sterilize by autoclaving
         SM may be stored indefinitely at room temperature.
10mM MgCl₂/10mM CaCl₂
0.2% maltose
10mM MgSO₄
Lambda top agar : 7g agar/L of culture medium
Chloroform
E.coli(DH5α)
YT medium
Chloroform, chloroform-isoamyl alchol (appendix 1)
PEG 8000
Phenol-chloroform

III. Experimental Procedures

1. Preparation of plating bacteria

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Place 50 ml of NZCYM supplemented with 0.2% maltose, and inoculate with a single bacterial colony. Grow culture overnight at 37℃．
Centrifugation the cell at 4000 rpm (Sorvall GSA) for 10 min at RT.
Resuspend pellet in 10 mM MgSO₄ (20 ml) and store the cell suspension at 4℃. The suspension may be used for up to 3 weeks But severely enfeebled strains of E. coli(e.g.,rec A^- strains) lose viability quickly when stored under conditions of starvation at 4℃. fresh culture of such strains sholud be used for plating.
 

2. Making a liquid lysate

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Grow an overnight culture of a lambda sensitive strain of E.coli in LB medium.
Pick a single plaque, blow it into a tube that contains 0.4 ml lambda dilution buffer, and place tube at 4℃ for 2 hr to allow phage to elute.
Combine 0.1 ml eluted phage with 0.1 ml of saturated culture and 0.1 ml of 10 mM MgCl₂ solution and incubate for 15 min in a 37℃ water bath.
Transfer this solution to 50 ml of NZCYM medium and shake vigorously at 37℃ until lysis occurs. (usually between 6-8 hr)
The culture should be checked frequently after 5.5 hr, and harvested immediately upon clearing.
Add a few drops of chloroform to lyse any remaining cells, transfer the solution to centrifuge tube, and spin for 10 min at 10.000rpm, 4℃.
Save as much of the lysate. Transfer to a screw cap tube, add a few drop of chloroform, vortex briefly, and store at 4℃.
Add DNase(10ug/ul) 2ul, RNase 2ul and incubate at 37℃ for 30min
Add DEPC treated DDW 2ul and then vortex.
 

3. Lambda phage preparation from liquid culture

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Add 30 ml of phage solution to 10% PEG/1.25 M NaCl(3g PEG + 2.2g NaCl) and vortex.
Incubate for 1 hr at 4℃ and then centrifuge for 30 min at 12000 rpm, 4℃. discard the supernatant, dry the inside of the tube and resuspended the phage pellet in 400 ul SM solution, Leave the sample overnight at 4℃ to allow the phage particles to dissipate.
 

4. Preparation of DNA from lambda phage.

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Add an equal vol. phenol-chloroform and rock gently for 5 min at RT.
Centrifuge at 12000 rpm for 5 min.
Transfer the aqueous phase to fresh tube and repeat step 2)-3) to three times. and then extract using 24:1 chloroform-isoamyl alcohol.
Transfer the aqueous phase to fresh tube. add 0.1 vol. of 3 M sodium acetate, and 2.5 vol. of ethanol, and gently rock the tube mix the contents.
Centrifuge at 12000 rpm 5 min.
Wash the pellet with 70% ethanol and dry the pellet.
Add 50 ul TE(pH 7.5), Dissolve DNA completely, and check the yield and quality of the DNA by recording a spectrum and electrophoresing.
 

5. Digestion of bacteriophage lambda DNA with restriction enzymes.

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Mix 20 ul of bacteriophage lambda DNA with 5 ul of the appropriate enzyme buffer and restriction enzyme.
Incubate for 1 hr at the optimal temperature recommended.
Agarose gel electrophoresis
 

IV. Notes

1. A plaque derives from infection of a single bacterium by a single virus particle.
2. Growth in maltose induces production in  E. coli of the lambda receptor (lamB protein), which is necessary for maltose transport. Mg⁺⁺ ions also aid phage absorption.
3. Lambda sensitive strains will support lytic growth. This can be tested by spotting 10 ul of a lambda lysate onto a lawn of bacteria. If the strain is lambda sensitive, a plaque will form where the phages were spotted.
4. If lysis does not occur or is incomplete, add an equal volume of prewarmed NZCYM or LB medium to the cultures and continue incubation for a further 2-3 hr at 37℃.
5. Although there can be considerable variability, stocks of bacteriophage lambda prepared from liquid cultures of infected bacteria or from confluent lysed plates contain between 10⁹ and 10¹⁰ pfu/ml
6. Aged batches of chloroform may inactivate bacteriophage lambda.
7. Typical yields of DNA isolated from the lambda phage contained on four 90 mm diameter confluent lawns is 0.2-0.5 mg, where as culture yields, for example, 2-5 mg λgt10 DNA from 1 L of E. coli. The latter method is time consuming, but the provision of several mg of DNA is useful when several attempts at subcloning are required.
8. Suspension of lambda phage can be directly for the preparation of DNA but the product is usually difficult or impossible to digest with restriction enzyme. this can be remedied to some extent by further purification through ion exchange columns.
9. A particle of bacteriophage lambda contains 5x10^-11 ug of DNA.
10. The background of plaques obtained after cleavage of bacteriophage lambda vectors at a single restriction site is often unacceptably high because of the accumulation of mutants in the original population that cannot be cleaved with the restriction enzyme. This problem can be avoided if the bacteriophage lambda vector is plaque purified frequently.
11. Simultaneous addition of PEG/NaCl works efficiently only if the bacteriophage grows well and the titer of bacteriophage in the original lysed culture is greater than 2x 10¹⁰ pfu/ml.
12. Particles of bacteriophage lambda are exceedingly sensitive to EDTA and other chelators, and it is essential that Mg⁺⁺(10-30 mM) be present at all stages of the purification to prevent disintegration of the particles.

V. References

Molecular cloning 2nd ed. Maniatis T. et al, CSH press 1989
Essential Molecular biology T. A. Brown IRL press 1991
Patterson, T. A. and M. Dean 1987. Nucleic acids Res. 15. 6298
Short protocols in molecular biology 2nd Ed. Ausubel F. M. et al Wiley 1992
Helms, C et al 1985. DNA 4. 29

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