Biology 212 Genetics Lab
Fall 2006
Lab 7: Transformation and Transduction in E. coli
Purpose:
To introduce genes into an E. coli strain by transformation and to transfer genes
from a wild type E. coli strain to a mutant E. coli strain by transduction with phage P1.
Introduction:
Transformation of E. coli
Transformation is defined as the heritable modification of the properties one
bacterial strain by the DNA of another. Since its discovery in Pneumococcus, genetic
transformation has been reported among several other bacteria such as Haemophilis
influenza and Bacillus subtilis. Early attempts to demonstrate transformation in E. coli
were unsuccessful. However, with the development of recombinant DNA technology,
several procedures have been worked out for the transformation of E. coli cells with
circular bacteriophage or plasmid DNA at high frequency. The procedure, known as the
calcium heat shock treatment, involves incubation of the cells in a hypotonic solution of
CaCl2 at 0-4C. This causes the cells to swell and become permeable to DNA. The DNA
which is added to this mixture forms a hydroxyl-calcium-phosphate complex which
adheres to the permeabilized cells. The DNA complex is absorbed into the cell by a brief
heat pulse. The cells are then grown in rich medium and can be plated on a selective
medium after about an hour.
The development of the calcium heat shock procedure for transforming E. coli has
played a major role in recombinant DNA research. It is now possible to examine the
physical properties of any foreign DNA fragment carried in E. coli. In this lab, the
plasmid pUC18 will be used to transform E. coli strain RR1. The pUC18 plasmid is a
circular DNA molecule. The plasmid contains genes for resistance to the antibiotic
ampicillin (ampR). A restriction endonuclease map is provided on the next page.
Transduction of E. coli
The transfer of genes by a phage into a host cell occurs by a process called
transduction. Once inside the host cell, the viral genome may enter a lytic or lysogenic
phase. In the lytic phase, the viral genome is actively replicated with concomitant
synthesis of the viral proteins and production of new viral particles. Intracellular
accumulation of newly produced viral particles eventually causes cell lysis. In the
lysogenic state, the viral genome is replicated at about the same rate as the host cell
genome, without the synthesis of viral proteins, and therefore without production of
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progeny phage particles. A viral genome in the lysogenic state is called a prophage. The
phage may integrate into the host cell genome by recombination at sites of sequence
homology. For some viruses, such as lambda, integration can only occur at a specific site.
Improper excision of integrated viruses can result in the transfer of adjacent genes by
specialized transduction. Generalized transduction is different from specialized
transduction in that essentially any bacterial gene can be transferred.
P1 is a generalized transducing phage of the bacterium Escherichia coli. During
the lytic cycle, the host cell genome is fragmented by virally synthesized endonucleases.
These small fragments are frequently incorporated into progeny viral genomes and
retained in the mature virion. When combined with appropriate selection, transduction by
P1 is a useful tool for constructing specific genetic characters in E. coli.
The strain you will transduce is E. coli RR1 (genotype: F- pro, leu, thi, lacY, Strr,
hsd-r, hsd-m). The genotype indicates that the RR1 strain is mutant in genes for proline
synthesis (pro) and leucine synthesis (leu), among others. The P1 lysate was made from a
wild type E. coli strain and therefore has normal proline and leucine genes packaged in
phage particles. You will attempt to repair the proline or leucine mutations in RR1 by
transduction.
The STANDARD PLATE COUNT is universally used to determine the number
of organisms in a bacterial culture. The procedure consists of diluting the organisms in a
series of tubes containing medium, as indicated below. Generally, three to four dilution
tubes are needed, but more can be used, if necessary. By using a dilution procedure
similar to the one below, a final dilution of 1:1 x 106 occurs in blank D. From blanks B,
C and D, measured amounts are transferred to plates containing appropriate growth
medium. The plates are incubated for 24 hours and examined. A plate which has
between 30 and 300 colonies is selected for counting. From the count, it is possible to
calculate the number of organisms per milliliter (ml) of the original culture. We will
carry out the plate count in order to be able to calculate a transduction frequency for the
introduction of pro+ and leu+ markers in E. coli RR1.
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Procedure:

Work in pairs. You should divide up the labor. One person can do most of the
transduction procedure, while the other does most of the transformation
procedure.

You should use proper sterile techniques throughout. If unfamiliar with sterile
procedures, ask your instructor to demonstrate them. Keep in mind:
o When using sterile pipets, don’t touch the ends or body of the pipets with your
fingers or lay the pipet down on the table before you use it.
o Use the pipet-aids, green (for 5 or 10 ml pipets) or blue (for 1 or 2 ml pipets),
to pump fluid into the pipet. Do not mouth pipet. Do not force glass pipets
into pipet aids—can result in injury.
o Pour off used medium and place used glass test tubes in dish pans containing
bleach. Used glass pipets should be placed in the labeled used pipet holders.
o Used plastic pipets should be discarded in the orange biohazard bags. Used
micropipet tips and microfuge tubes should be discarded in the beakers for
biohazard waste on the benches.
Transformation
1. Obtain 2 sterile microfuge tubes and place on ice. One tube should be labeled “control
and one “amp”.
2. Add 0.1 ml of competent RR1 cells (in tubes on ice—do not use the bacteria from the
flasks). Competent cells have already been treated with Ca2+ to make them able to take
up DNA to each of the tubes.
3. To the control tube, add 10 l of TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA) using a
micropipet. To the “amp” tube, add 10 l of 40 ng/l pUC18 DNA (or other plasmid
DNA). Let the tubes stand on ice for 10 min.
4. After the cells and DNA have been incubated on ice, place the tubes in a rack in a
37C water bath for 5 min. Place the tubes back on ice.
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5. To enable expression of the protein encoded by the ampR gene, add 1 ml of sterile LB
medium to the transformation mixtures. Return to the 37C water bath for 30 min.
6. While you are waiting, label two LB + ampicillin plates on the back (agar) side. Use a
permanent marker to label “control” and “amp” plates and include your name or initials.
7. Carefully place 0.1 ml of the cells marked “control” on the plate labeled “control”,
using a sterile pipet. Also sterilely place 0.1 ml of cells marked “amp” on the plate
labeled “amp”.
8. Spread the cells evenly over the plates with a sterile glass spreader (flamed in ethanol
and cooled before spreading—the instructor or TA will demonstrate). Incubate the plates
inverted at 37C overnight. The instructor will remove the plates for the incubator and
store them until next week’s lab.
9. (Next week) Record the number of transformants on each plate.
Transduction
1. You will be given a culture flask containing E. coli RR1 in high exponential growth.
Using a sterile pipet, transfer 5 ml to a sterile test tube. Add 1 drop of 0.5 M CaCl2. You
should start the 30 min. incubation for step 3 below first, then complete the dilution
and plating steps 2 a-m.
2. Quantitative plating method. a. Transfer 0.1 ml of cells from step 1 to 9.9 ml of LB
(Blank A) in a large test tube (1:100 dilution).
b. Vortex Blank A well.
c. Transfer 0.1 ml from Blank A to 9.9 ml of LB (Blank B) in a large test tube (1:1 x 104
dilution).
d. Vortex Blank B well.
e. Transfer 1.0 ml from Blank B to 9.0 ml of LB (Blank C) in a large test tube (1:1 x 105
dilution).
f. Vortex as above.
g. Transfer 1.0 ml from Blank C to 9.0 ml of LB (Blank D) in a large test tube (1:1 x 106
dilution). Vortex as above.
h. Transfer 0.1 ml from Blank B to an LB plate marked 1:10,000. Spread cells as in
transformation procedure.
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i. Transfer 0.1 ml from Blank C to an LB plate marked 1:100,000. Spread cells as above.
j. Transfer 0.1 ml from Blank D to LB plate marked 1:1,000000. Spread cells as above.
k. Incubate plates, inverted at 37C overnight. Make sure plates are properly labeled.
Instructor will remove plates the next day and store in a refrigerator.
l. (Next week) Lay out plates from transduction step 2 on a table in order of dilution.
Select the plate that has no fewer than 30 nor more than 300 colonies.
m. (Next week) Calculate the number of cells/ml of culture by multiplying the number of
colonies counted by the dilution factor and dividing by the volume of cells plated.
3. Transduction of RR1 with P1 lysate. Remove 0.5 ml of cells from step 1 and place
them into a sterile eppendorf tube. Add 2 drops of P1 lysate. Let sit undisturbed at room
temperature for 30 min.
4. Centrifuge the cells for 2 min. at 10,000 rpm in a microcentrifuge. Use a sterile pipet
to carefully remove all the supernatant, which should be discarded in a dishpan with
bleach. Add 0.5 ml of glucose minimal medium to the pellet in the tube, cap the tube and
resuspend the cells by vortexing.
5. Transfer 0.1 ml of transduced cells onto a minimal plate containing leucine (selection
for pro+) and 0.1 ml onto a minimal plate containing proline (selection for leu+). Spread
cells over the surface of the plate using a sterile glass rod.
6. As a control for revertants (back mutations leu- --> leu+ or pro- ---> pro+; these are
RR1 cells whose genotype is altered by mutation rather than transduction) remove 0.5 ml
of cells from step 1 and place them in a sterile microfuge tube. Centrifuge the cells for 2
min. at 10,000 rpm in the microcentrifuge. Remove the supernatant with a pasteur pipet
and discard the supernatant in a bucket of disinfectant. Add 0.5 ml of glucose minimal
medium to the pellet in the tube, cap the tube and resuspend the cells by vortexing.
7. Spread 0.1 ml of control cells from step 6 onto a minimal plate containing leucine and
0.1 ml onto a minimal plate containing proline.
8. Incubate the plates, inverted at 37C for about 48 hours. Make sure plates are labeled.
Instructor will remove plates and store in refrigerator.
9. (Next week) Record the number of transductants you observe on each plate. Note that
leu+ transductants will grow on the plate to which proline was added; pro+ transductants
will grow on the plate to which leucine was added. Also note whether any revertants
were observed on the control plates. Revertants are due to a new mutation that alters the
growth phenotype in the absence of added phage.
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BIOL 212 Genetics Lab
Transformation and Transduction of E. coli
Name___________________
Section_________________
Assignment: Worksheet. Analyze your results from this lab, between Thursday of this
week and Tuesday of next week (the next lab period). Answer the questions on the
worksheet to be turned in by the end of the next lab period.
1. Were you successful in transforming the E. coli RR1 strain with the plasmid DNA? If
unsuccessful, analyze other experimental plates with transformed colonies. Describe your
results (count the number of colonies on tranformation and control plates).
2. Transformation frequencies are often expressed as number of ampicillin resistant
transformants/g of tranforming DNA. Explain how you could calculate the
transformation frequencies for ampR colonies from your results. Present your raw data
and show your work to calculate the frequencies.
3. What is the number of cells/ml of original E. coli RR1 culture as determined from
steps 2a-m of the transduction procedure? Show your work.
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4. How many transductants did you observe on each plate? Complete the table below:
Table 1: Raw data from transduction lab
marker gene
transductants
experimental
pro+
leu+
marker gene
revertants
control
pro+
leu+
5. Determine the transduction frequency for pro+ and leu+. To exclude revertants,
subtract the number of colonies observed on the control plates from the number on the
experimental plates. Express in terms of number of pro+ or leu+ transductants per number
of input cells. Show your work.
6. Using either transduction or transformation, design an experiment to convert thimutant E. coli cells to thi+ protrophic cells. Thi- mutants require the vitamin B1
(thiamine) for growth. Outline important steps of the procedure to be carried out. Be sure
to include appropriate controls and explain what selective media would be used to
distinguish the thi+ cells from the original thi- cells.
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