PROCEDURES FOR TRANSFORMATION EXPERIMENTS

Preparation of Media:

H.I. grows well in a Levinthal medium, The medium used in this laboratory
has been modified slightly from the original description. It is prepared the
following way:

(1) 37 gms. brain heart infusion media in 1000 liters of distilled H20

bretight to a boil,

(2) 100 ml. definbrinated sheeps blood added a little at a time to prevent
excess frothing. |

(3) Allow mixture to settle one or two minutes, then filter through a fluted
filter (Whatman #12 ).

(li) Refilter gamer 50 ml. of the filtrate. jet

(s) «fdrclowe. foe's 20-28! (oe longer)

(6) This constitutes Levinthal stock and is used 1:1 with sterile Eugonbroth

 

as Levinthal broth (B.B.L. Eugonbroth 30 gms.,;l. of HO) .

(7} For plates aad #% agar to Eugonbroth, sterilze in autoclave 20 mins.
at 120°C. and upon cooling to 45-50°C. mix 1:1 with Levinthal stock
(45°C) for Levinthal agar ({#%).

(8) Levinthal stock is usually kept in 200 ml. quantities and mixed with
200 ml? 3 Eugonbroth for platings. Levinthal stock is kept in 50 ml,
quantities and mixed with 50 ml, of Eugonbroth for use as a liquid growth

medium.

(4) Too) ¥ fk PPM hei) 0 Aad C hb nadir kif wae.

Dilutions:

(1) Dilutions are always made with Eugonbroth or some ciluent containing
protein,
(2) 2x 102 dilutions are usually made with 9.9 ml. of broth and 10+ dilutions

with 9 ml. of broth.
Procedures for Transformation Experiments (Cont.)

Platings:
(1) 20 ml. of media per plate is normally used for pour plates.

(2) If an agar layer technique is employed, two 10 ml. layers are used.

Handling of T.P.:
(1) Always dilute transformation preparations (T.P,) in saline-citrate buffer
(.15 M NaCl + .014 M Na citrate).

(2) Dilute preparations of T,P. should be stored frogen,::

Gels:
Transformations depend to a large extent upon the phsyiological state

of the organism at the time at which DNA is added to the cells. Cells to

be transformed are shaken gently for aeration--not vigorously since the cells

are broken up by this process-aat 35-37°C. to give a concentration of
eee

approximately, 34-207" organisms/ml., (Coleman reading of yy i

adapeer>.

~~)
TRANSFORMATION EXPERIMENTS

One may define transformation as the transfer of genetic characteristics
to a host cell by means of DNA prepared from a donor population, Transforma-
tions have been described for ¢segorganisms and perhaps one or two more

questionable cases, Hemophilus influenza (H.I.) will be used to demonstrate

 

transformation in the experiments to be disdéussed below,

DEMONSTRATION OF TRANSFORMATION AND TITRATION CURVE OF & TRANSFORMING FAR

One of the easiest methods of demonstrating transforming activity utilizes
the characteristic of streptomycin resistance, Rough H.I. type d, or simply
Hi. Rd, or Rd is suceptible to Ie gamma/ml, or less of streptomycin while
the mutant "strep resistent" is capable of growth in media containing greater
than 2000 gamma/ml. of streptomycin, DNA prepared from cells resistant to
streptomycin transform susceptible cells to streptomycin resistence with a
frequency 6 or more orders of magnitude greater than the spontaneous mtation
frequency.

In this experiment also the frequency of transformation will be related
to the concentration of DNA added, i.e., serial dilutions of transforming
principle (T.P.) will be added to a constant number of cells and the frequency
of transformation determined,

Experiment XXII
(1) To 7 test tubes (size 22 x 175 mm) add/.8 ml. of Levinthal broth. Number
the tubes 1-7. |
(2) To tube #1 add «1 ml. of saline buffer, To tube #2 add .1 ml. of 10°
dilution of T.P,, to #3 add .1 ml. of 103 dilution of T.P., etc. To
tube #7 add .1 ml. of 10% dilution of T.P. and add .05 ml. of a DNAse

preparation calculated to give 1 gamma/ml.

/3
(3)

(4)
(5)

(6)
(7)

(8)
(9)

(10)

After 1 minute add to each tube .1 ml. of H.I. cells, grewn—from an
inoculum_of_1x-108_ceprefntr—to-approximatety—ir5-—2-20%¢mt—tthe
Instructor will provide celis).

Incubate tubes for 20 minutes at 35-37°C, with gentle shaking.
Plate cells according to the following schedule with 10 ml. of

Levinthal agar cooled to 37-0°C:

Tube Dilution ( Suede digenta on come Mivcs 4 eiti-)

1 Undiluted
2&3 102

hy 101

5 100

6 10°

Incubate plates 2 hrs. at 35-37°C.

Layer plates with 10 ml. of Levinthal agar containing 500 gamma/ml. of
streptomycin. Incubate at 35-37°C.

At the next period count the number of colonies per plate.

Plot the number of colonies transformed in each tube as a function of
DNA dilution,

What significance can be attributed to the shape of the curve.

If you assume that 10 per cent of the DNA in the T.P. was taken up by

ike e@ells and the molecular weight of the DNA and T.P. is 6 x 10°, then

calculate the ratio of T.P. molecules to DNA molecules.

/35
ISOLATION OF T.P, BY THE LYSOZYME TECHNIQUE AND CALCULATION OF T.P. PER DONOR CELL.

Experiment XXIII

This experiment illustrates the fact that the T.P. introduced into the

cell is transmitted by cell growth as genetic material, i.e., it is not only

responsible for the streptomycin resistence, but it also replicates,

(1)

(2)

(3)

(h)
(5)

Sa) Heat ose
Ins? 76)
(7)
(8)

Isolate a colony of H.I. transformed to streptomycin resistant: ig Oval.
of Levinthal broth and grow culture to a turbid suspension, This operation
will be performed by the instructor in cases of conflict of time since

it will be necessary to have the cells available at the start of the
period,

Dilute cells 1/3 with Levinthal broth, 1.2 ml, cells + 2,4) ml. broth.

Make a viable cell count at a dilution of 2x 107,

lyse wv
To, 3 ml, of suspension add .1 ml. of lysozyme (final concentration to a He

necked .
100 gamma/ml) and shake at 37°C for 10 minutes, “

,Add .2 ml. of 1 N NaOH. This should produce a pH of about 10,3,

When the cells lyse as judged by a clearing of the suspension--usually
1 minute is sufficient--neutralize the NaOH by adding .2 ml. of 1 N HCl.
This material is a crude preparation containing T.P. activity for
streptomycin resistence and will be used to transform Rd cells,
Dilute crude T.P, 103 with citrate-saline.
To 3 tubes addj,8 ml. Levinthal broth + .1 ml. of crude T.P. (103 dil.).
Number these tubes 1-3. To Tube #1 add .05 ml. DNAse (1 gamma).

To tube #2 add .05 ml. RNAse (10 gamma).

To tube #3 add ,05 ml. saline buffer.
To each tube add .1 ml. of Rd cells, grown-te-e-ecrreenteation of

ee $55 -x-t09—tarrvittty—s—r20) .

/%
(9) Incubate cells at 35-37° for 20 minutes and plate at 10° dilution
+2710 ml. of Levinthal agar, |
(10) As a control plate .1 ml. of T.P. (105 dil.).
(11) Incubate plates 2 hrs. at 35-37°C, and layer plates with 10 ml. of
Levinthal agar with 500 gamma/ml. of streptomycin, |

(12) Incubate at 35-37°C. and count at the next period,

Assuming no variation in transformation from Experiment XXII calculate
the amount of DNA-T.P. present in the lysozymed culture from the curve
obtained in Experiment XXII, given the value of 200 gamma/ml. of DNA for
the T.P, (undiluted) used in the previous experiment.

The amount of T.P. taken up by the cells may be obtained by centrifuging
the cells and then titering the supernatant for loss of T.P..Assuming a value
of 10% uptake of T.P. by the cells in this experiment, calculate the number

of units of T.P. contrifuzed per lysozymed cell.

/37
Experiment XXIV

DNA prepared from various sources will interfere with transforming DNA
if present in sufficiently high concentration. In this experiment DNA
extracted from host and donor cells will be used to test for direct interference,
The T.P. used will be T.P. obtained from streptomycin resistant cells, inter-
fering DNA will have come from Rd streptomycin sensitive cells,

(1) To h tubes add/.7 ml, Levinthal broth + .1 ml. of T.P. diluted 102.

Number tubes 1-l, To tube #1 add .1 ml. saline buffer.

To tube #2 add .1 ml. DNA 101 dilution.
To tube #3 add .1 ml. DNA 10? dilution.
To tube # add .1 ml. DNA 103 dilution.

To an empty tabe (#5) add .8 ml. Levinthal broth + .1 ml. of DNA 104

dilution. To another tube (#6) add .7 ml. Levinthal broth +2 ml. of

DNA 10? dilution,

(2) To each tube add .1 ml, Rd Ctudcebes—m— 96>,
(3) Shake for 20 mimtes at 35-37°C. However, at 2 min. add .1 ml. of 102

dilution of T.P, to tube #6.

(4) Plate cells according to the following schedule:

#1 - 102 #h = 102
#2 ~ 101 #5 - 109 .
#3 - 102 #6 ~ 1ol

(5) Incubate plates at 35-37°C. for 2 hours and layer with streptomycin

agar containing 500 gamma/ml of streptomycin. Incubate,

From the previous experiment you calculated that T.P. preparations from
Sd cells consist of 1T.P. molecule for every 350 molecules of DNA of 6 x 106
molecular weight. Assuming this to be true, can you arrive at a figure from
the above experiment for the number of DNA molecules that interfere With T.P,
uptake by cells?
/38