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Protocol 1.2: Making a Phage Stock—Growing λ-Tn5'

Protocol 1.2: Making a Phage Stock—Growing λ-Tn5'

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TRANSPOSON MUTAGENESIS OF Escherichia coli



9 E. coli strain that contains a nonsense suppressor and can support the



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lytic growth of )~::Tn5'~BW11397

)~ Ym m e d i u m

sterile test tubes

spectrophotometer to monitor bacterial cell density, such as Spectronic

20, or Klett meter

)~ agar plates poured thick, within the last 5 days

water bath or heating block at 45-50~

Bunsen burner and matches

0.1-, 1-, 5-, and 10-ml sterile pipets in canisters

pipet aids

sterile glass capillary pipets and appropriate pipet aid



Procedure

1. Inoculate an E. coli nonsense suppressor strain (BWl1397) into 5 ml

of ~ Ym broth; incubate culture overnight with shaking at 37~

2. The next day, start a culture of the E. coli nonsense suppressor strain

(BWl1397) in ~ Ym broth using an inoculum from the overnight culture.

Grow the E. coli culture to mid-log phase. This is an optical density of

approximately 0.6 at 660 nm in a spectrophotometer or approximately

100-120 Klett units in a Klett meter. For example, 0.1 ml of an overnight

culture diluted into 2.5 ml of X Ym broth will grow to mid-log phase

in about 3 hr with a vigorous shaking at 37~

3. Melt ~ top or soft agar in a boiling H20 bath or a microwave. Place

the melted top agar in a heating block or water bath at 45-50~ to

COO1.



4. Make serial dilutions of the )~phage TnphoAlacZ-132(4253, also called

TnphoA'-2) in )~ Ym broth. Prepare to plate phage dilutions that will

give 50 to 100 plaques per plate.

5. Transfer 0.1 m] of each phage dilution to be plated to appropriately

labeled sterile test tubes. Add 0.1 m] of the mid-]og phase bacteria]

culture to each tube.

6. Mix and incubate the a]iquots of the phage dilutions with bacteria at

room temperature for 20 min to allow time for phage to adsorb to

bacteria.

7. To each tube, add 2.5 m] of melted )~ top agar, coo]e(] to 45-50~

Quickly over]ay the mixture onto fresh )~ agar p]ates. These plates

should have been poured within the last 5 days and should be very

thick (i.e., ~2x usual volume of medium per plate). (Phage plaques

wi]] grow larger on very fresh plates with a high moisture content.) Let

the overlay solidify completely before moving the plates.



PROTOCOL 1.2: MAKING A PHAGE STOCK--GROWING ,-Tn5'



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8. Incubate the plates inverted at 37~ overnight.

9. The next day do Part B.



Part B: Making Phage Lysate Stocks

The plates prepared in Part A the day before are used to amplify

individual plaques to make phage lysate stocks.



Materials

9 plates with individual plaques of phage prepared in Part A the day

before

9 overnight culture of E. coli strain that contains a nonsense suppressor

and can support the lytic growth of )~::Tn5'--BW11397 grown in k Ym

broth

9 Tris phage buffer

9 sterile Pasteur pipets

9 pipet bulbs or pipet aids

9 sterile test tubes

9 k top agar

9 k agar plates poured within the last 24 hr

9 water bath or heating block at 45-50~

9 Bunsen burner and matches

9 0.1-, 1-, 5-, 10-ml sterile pipets in canisters

9 pipet aids

9 sterile glass capillary pipets and appropriate pipet aid

9 clinical centrifuge or preparative centrifuge

9 sterile centrifuge tubes for the above centrifuge

9 chloroform



Procedure

Begin with phage plates prepared in Part A the day before. Several

different phage lysates, five for example, are prepared.

For each phage lysate:

1. For each lysate to be made, add 0.1 ml of Tris phage buffer to each

test tube. Use a sterile Pasteur pipet and a pipet bulb to "core out"

or pick up a plaque. Place a well-isolated individual plaque or several

individual plaques from the overnight plates in Part A in the tube.

Select )~ plaques that are "average looking," not extreme in size or

morphology. The k plaques can be "cored out" and picked up using

a sterile Pasteur pipet. The use of very fresh plates helps accentuate

the differences in plaque morphologies.



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TRANSPOSON MUTAGENESIS OF Escherichia coli



2. Allow the phage plaque to incubate in the Tris phage buffer for 30

min at room temperature.

3. Melt )~ top agar in a boiling H20 bath or a microwave. Place the

melted top agar in a heating block or water bath at 45-50~ to

COO1.

Note: For this step, use top agar that has been melted only once. For

phage titers, top agar melted several times may be used.

4. Add 0.1 ml of an overnight culture of bacteria (BW11397) in )~ Ym

medium to the test tube. Mix. Adsorb phage to bacteria for 20 min at

room temperature.

5. Add 3 ml of freshly melted top agar, cooled to 45-50~ to the test

tube.

6. Pour onto a very fresh )~ agar plate. Use plates poured within the last

24 hr or less (poured with -~25 ml agar per plate).

7. Be sure to make a control plate (bacteria alone, no phage).

8. Incubate at 37~ for 6 to 8 hr until there is evidence of lysis on the

plate. The plates should look "lacy" or almost clear because there are

many plaques very close together. The no-phage control plate, which

should have a solid lawn of bacteria, is very helpful for comparison

to determine when the phage-containing plates have lysed or cleared.

9. After the plates have cleared, flood each agar plate with 5 ml of Tris

phage buffer. Store the plates in the refrigerator for 12 to 24 hr. Be

careful not to tip flooded plates.

10. After 12 to 24 hr, tilt each plate and remove the buffer containing the

phage lysate using a sterile Pasteur pipet or other glass pipet. Place

the phage suspension in a screw-capped centrifuge tube.

11. Add a few drops of chloroform to the tube and cap securely. Mix by

inversion.



CAUTION: Wear gloves and protective goggles when handling chloroform. DO NOT mouth pipet chloroform. Keep chloroform away from heat

and open flames.

12. Spin the sample for 10 min in a clinical centrifuge at 3000 rpm or in

a preparative centrifuge for 5 min at 5000 rpm.

13. Carefully remove the supernatant solution with a pipet and transfer

the supernatant fluid to a new screw-capped centrifuge tube. Do not

remove the debris or chloroform.

14. Add a few drops of chloroform to the supernatant, mix, and centrifuge

again. Carefully remove the supernatant solution to a new tube. Add

a few drops of chloroform to the tube. Label the tube clearly. This is

a )~::TnphoA'-2 lysate. Store at 4~

15. Determine the titer of the phage lysate using Protocol 1.1.



PROTOCOL 1.3: TRANSPOSON MUTAGENESIS USING



,~::TnphoA'-2



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NOTES



1. The phage lysates may be stored at 4~ indefinitely. Phage titers of 10 9

to 101~ a r e typically obtained. In step 8, if it is inconvenient to check

plates for lysis after 6 to 8 hr, the plates can be prepared in the afternoon

and incubated overnight. This may reduce the phage titer somewhat,

but still gives satisfactory results.

2. If there is not enough time to allow the phage to diffuse from the top

agar into the buffer, phage can also be removed from top agar by scraping

the top agar off a plate and into a centrifuge tube. Add 5 ml of Tris phage

buffer and 0.1 ml of chloroform. Vortex to mix thoroughly. Centrifuge 5

min at 10,000 rpm. The supernatant solution is the phage lysate.



PROTOCOL 1.3:

Transposon Mutagenesis Using ~::TnphoA'-2

A transposon can be introduced into E. coli cells to be mutagenized

by the infection process of bacteriophage )~. The E. coli strain to be mutagenized must be sup § so the phage containing amber mutations in the O

and P genes needed for lytic DNA replication of ~ cannot grow lytically

in the strain. The host strain must be tetracycline sensitive so the E. coli

cells with a transposon that renders them tetracycline resistant can be

selected.

Materials

9 E. coli strain to be mutagenized, freshly grown on an L agar plate the



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day before this procedure (BW10748 that is nonpermissive for )t phage

replication)

Phage lysate stock of )~ phage TnphoAlacZo132

L broth + 0.4% maltose

MC buffer (10 mM MgC12, 10 mM CaC12)

sterile test tubes

shaking water bath at 37~

L t e t l 5 agar plates (15/zg/ml tetracycline)

bent glass rod and beaker with alcohol to spread bacterial cells on agar

plates

clinical or preparative centrifuge

sterile centrifuge tubes



Procedure

1. The day before use, streak the bacterial strain (BW10748) to be mutagenized on an L agar plate and incubate overnight at 37~



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TRANSPOSON MUTAGENESIS OF Escherichia coli



2. The day of this procedure, inoculate a single colony of BW10748 into

5 ml of L broth + 0.4% maltose.

3. Grow the culture 4 to 6 hr with shaking at 37~ The bacterial culture

will be in mid- to late log phase growth at that time.

4. Harvest the bacterial cells by centrifugation for 5 min at 3000 rpm

in a clinical centrifuge or for 5 min at 5000 rpm in a preparative

centrifuge.

5. Decant the medium and resuspend the cell pellet in I ml of MC buffer.

6. Distribute 0.1 ml of resuspended cells to each of several small sterile

test tubes.

7. Add to the tube a series of different phage concentrations. Make dilutions in MC buffer. Prepare a range of tubes similar to those suggested.

To tube 1 add 10 ~1 of the phage lysate.

To tube 2 add 10/~1 of a 1:10 dilution of the phage lysate.

To tube 3 add 10 ml of a 1:100 dilution of the phage lysate.

To tube 4 add nothing.

8. Incubate 20 min at room temperature.

9. After adsorption, spread the cells and phage or dilutions of the cells

and phage o n Ltetl 5 agar plates (15/~g/ml tetracycline). To spread the

cells on the agar plate, sterilize a bent glass rod by dipping it in a

beaker of ethanol. Drain excess ethanol off the glass rod. Briefly pass

the rod through a Bunsen burner flame. When all the ethanol has

burned off, touch the glass rod to the inside of the petri plate top to

cool it. Use the glass rod to spread the aliquot of cells evenly on the

agar plate.

10. Incubate at 40~ overnight.

NOTES



1. The E. coli strain to be mutagenized is grown in medium containing

maltose because growth in maltose in the absence of glucose induces

the formation of more )~ phage receptors on the surface of the E. coli

cells. The receptor for bacteriophage )~ binding and uptake into the E.

coli cell, encoded by the lamb gene, is also the channel for maltose

uptake into the E. coli cell. Growth of E. coli in the presence of maltose

induces the formation of more receptors for maltose uptake on the

surface of the E. coli cell. Maltose is broken down to glucose in the E.

coli cell. Growth of E. coli in the presence of glucose prevents the

induction of more maltose receptors on the cell because of catabolite

repression.

2. An alternative way to prepare the E. coli cells to be mutagenized is to



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