EXPERIMENT 2: Polymer Synthesis after Infection

Bacteria as noted in Experiment 1.

Bacteriophage as noted in Experiment 1.

Media

For MS2, TPG medium:

KCl 0.11 M KH,PO,, 1.0 mM

NaCl 0.008 M Na pyruvate 0.073 M

NH,CL 0.05 M FeCl, 2 uM

Tris 0.10 M CaCly 0.1 mM

Bring to pH 7.4 with HCL MgCl5 1.0 aM

NajS0, 0.16 mM Glucose 0.2 gm/L
For 14, M-9 medium:

NH,C1 1.0 gm

MgSO, 0.13 gm

KH9P0, 3.0 gm
NaoHPO, 6.0 gm
Glucose 4.0 gm
Water 1 liter

For T4 infection, add L-tryptophan, 20 mg.

The purpose of this experiment is to measure nucleic acid and protein
synthesis in cells infected with normal T4 or MS2 and amber mutants of
these phages. In the case of MS2, E. coli will be treated with actinomy-
cin D prior to infection in order to inhibit host RNA and protein synthe-
sis. Each pair of students will use a specific phage as indicated below:

A. T4 series

1. Uninfected B

2. Tar in B

3. T4& aml22 in B
4. T4 aml02 in B
5. T4& amBl7 in B
6. T4r in B

B. MS2 series

- MS2 in C3000
2. Uninfected C3000
3. MS2 am2 in C3000
4. MS2 am9 in C3000
5. MS2 ts4 in C3000
6. MS2 in C3000
To be supplied:

1. Bin M-9 medium at 2 x 10?/ml

2. ©3000 in Tris HCl at 4 x 109/ml

3. Phage at 1019 pfu/mi (T4) or 4 x 109/mi (MS2)

4. 13 x 100 mm tubes containing H2-thymidine (T), H3-uridine (U)

or C14-valine (A)

5. Filter paper discs, pins and foam rubber pin cushion
6. TPG-actinomycin (5.5 ug/ml actinomycin)

7. Thymidine-uridine-amino acid mixture (TUA mix)

8. EDTA-Tris

9. 5% TCA
10. 10% TCA
11. Alcohol-ether (1:1 by volume)
12. Ether
13. Scintillation counter vials
14. 100 uL disposable pipettes

Before the experiment begins, prepare filter paper discs for samples
as demonstrated.

Protocol for T4 -- Period 1

-2' - Pipette 0.2 ml of B into a 13 x 100 mm tube in 30° water bath.

0' = Add 0.2 ml of phage (2 x 10? pfu) or in case of uninfected con-
trol, 0.2 ml of nutrient broth.

4' ~ Add 2.0 ml of M-9 medium

5' = Distribute 0.6 ml to each isotope tube at 30° (T, u,@;).
Leave the remaining culture at 30° (Tube 4). Shake all tubes.

7' + Remove 0.1 ml from each isotope tube (T, U,\A;) with a separate
capillary pipette and deliver onto filter paper discs marked
T7, U7,‘A)7, respectively. (Remember the sample is radioactive!)
Immediately after applying sample to disc, add 1 drop “TUA mix"
to quench (use a Pasteur pipette). (Keep capillary pipettes
in empty 13 x 100 mm tubes for further use. Do not mix up T,
U, A pipettes.)

10' - Dilute 0.1 ml from tube 4 to 1073 and 1074 and spread 0.1 ml
on T4 plates.

15')

30')- Repeat sampling as at 7'

60')

Leave all tubes in bath. Store discs in your locker.

Protocol for MS2 -~ Period 2

-10'- Heat 0.2 ml EDTA-Tris to 41° in a can of tap water.
-8' - Add 0.2 ml C3000 and keep at 41°
-5' - Add 0.2 ml of EDTA-treated cells to 2 ml TPG-actinomycin,
which has been shaking at 37° in a 18 x 150 mm tube.
0' - Infect cells at multiplicity of 5 (total of 2 x 109 pfu)
Controls: do not add phage
-3-

S5' - Distribute 0.6 ml of cells to each of two tubes labelled Ur
(for H3-uridine) and A (for Cl4-amino acids).

10' - Withdraw 100 pl from Ur and from A with capillary pipettes and
apply to Ur-10 and A-10 discs, respectively. Immediately add
1 drop of TUA mix to each disc with a Pasteur pipette. Keep
pipettes in clean tubes (Ur or A) for further use.

20°)

30')- Repeat sampling

60')

Leave all tubes in water bath: Store discs in your locker.

Washing filter paper discs -- Period 3
Put all T, U, Ur discs in one 250 ml beaker, leaving pins in the

discs. Do not touch discs with fingers; handle the pins only. This is
beaker 1.

Put all the A andiA'discs in a second 250 ml beaker. This is beaker 2.

 

Label each beaker with your pair number.

Keep beaker 1 in an ice bath, add ice-cold 5% trichloracetic acid (ICA)
to cover discs, and allow to soak for 10 minutes. Decant the TCA care-
fully (radioactive!) and repeat 4 more times for 10' periods. Wash discs
twice with alcohol-ether for 5 minutes, twice with ether, and allow to dry.

To beaker 2 add cold 10% TCA and allow discs to soak for 30 minutes.
Decant carefully (radioactive!) and add 5% TCA. Heat at 90° in a pan
of water for 15 minutes. Wash the discs three times with 5% TCA for 5

minutes at room temperature and then with alcohol-ether and ether as
above.

Place discs without pins in scintillation vials using gloved hand,
and label tops with pair number and disc number. Instructors will
collect and count your vials in a liquid scintillation counter. When
the vials are counted, the results will be returned to you. Plot your
results and compare them with those of others.