Bacterial Transduction GeNeiTM Bacterial Transduction GeNeiTM

GeNeiTM Bacterial
Transduction
Teaching Kit
Manual

Cat No. 6112300011730

Revision No.:00050516
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Bacterial Transduction GeNeiTM Bacterial Transduction GeNeiTM

CONTENTS

Page No.

v Objective 1

v Principle 1

v Kit Description 3

v Materials Provided 6

v Procedure 7

v Observation & Interpretation 12

v Appendix 13

v Ordering Information 14

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Bacterial Transduction GeNeiTM Bacterial Transduction GeNeiTM

Objectives:
To understand the process of Bacterial Transduction through
the lytic and lysogenic life cycle of bacteriophage, by
performing.
• Bacteriophage infection
• Lysogenization
• Temperature induction of bacteriophage (lytic cycle)
• Selection of transductants and
• Phage titration

Principle:
Genetic recombination is the transfer of DNA from one
organism to another. The transferred donor DNA may then
be integrated into the recipient's nucleoid by various
mechanisms and it confers a new property to the recipient
cell like antibiotic resistance or production of some amino
acids etc
Natural mechanism of genetic recombination in bacteria
include
• Transformation
• Transduction
• Conjugation
Transduction is the transfer of fragments of DNA from one
bacterium to another bacterium by a bacteriophage. This
genetic transfer occurs in both Gram positive and Gram
negative bacteria.
Transduction is observed with temperate bacteriophage (those
that can form prophages) A prophage is a bacterial virus that
has integrated its DNA into the DNA of a bacterial cell. This
process of integration of viral DNA into bacterial DNA is called
lysogenization.

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Bacterial Transduction GeNeiTM
There are 2 types of transduction
1.Generalized transduction
2. Specialized transduction
Generalized transduction: In this process, any part of the
bacterial chromosome can be transduced. i.e as the phage
begins the lytic cycle, bacteriophage enzymes hydrolyse
the bacterial chromosome into many small pieces of DNA.
Any part of the bacterial chromosome may be incorporated
into the phage head during the phage assembly. Generalized
transduction is said to occur when the phage has a roughly
equal chance of carrying any segment of the donor's
chromosome.
For Ex. Bacteriophage P1 in E.coli, P22 in Salmonella
Specialized transduction: In this process, only certain
pieces of the bacterial chromosome can be transduced. More
specifically the phages transduce only those bacterial genes
adjacent to the prophage in the bacterial chromosome. Thus
the process is also called restricted transduction. It occurs
when a bacteriophage genome, after becoming integrated
as prophage in the DNA of the host bacterium, again becomes
free upon induction and takes with it into the phage head a
small adjacent piece of the bacterial chromosome. In this
way, when such a phage infects a cell, it carries with it the
group of bacterial genes that has become part of it. Such
genes can then recombine with the homologous DNA of the
infected cell. Specialised transduction occurs when a gene
or a set of genes has a high frequency of transduction relative
to the majority of genes on the bacterial chromosome.
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Bacterial Transduction GeNeiTM
Kit description:
In this kit, an E.coli phage is supplied, which exhibits both
lytic and lysogenic life cycle. Three E.coli strains (Donor,
Recipient and Susceptible host) are provided in the lyophilized
forms.
This kit simulates the process of transduction: i.e. genetic
transfer of antibiotic resistant gene from one E.coli (Donor)
strain to another (Recipient) through the bacteriophage.
The donor strain is resistant to chloramphenicol and the
recipient is sensitive to this antibiotic but resistant to
ampicillin. Upon phage infection, phage DNA enters the donor
cell and integrates into the bacterial chromosome. On
temperature induction, the phage DNA is excised from the
bacterial chromosome, the phage replicates and the host
cell lyses releasing mature phage particles. These phage
particles are then taken up for infecting the recipient strain
(chloramphenicol sensitive) where lysogenization occurs. The
screening of the transductants is based on the antibiotic
selection marker. The recipient strain which will acquire the
chloramphenicol resistant gene from the phage will survive
on LB chloramphenicol plate, thus indicating the transfer of
the gene from donor to recipient through a bacteriophage. To
confirm the presence of the phage in lysogenized recipient
strain, the phage is induced and titrated against the given
susceptible host.
3
Bacterial Transduction GeNeiTM Bacterial Transduction GeNeiTM

6112300011730 : The kit is designed to carry out

5 tranduction experiments using single
lyophilized vials of E.coli strains (3 Nos.
Donor, Receipient and Susceptible
Host).

Duration of Experiment: Experiment is carried out over a

span of 4 days. Approximate time taken on each day is
indicated below:
Day 1 : 2 hours (preparation of media and revival of host)
Day 2 : 8 hours (inoculation, induction followed by a spin
and filtration)
Day 3 : 6 hours (incubation and plating)
Day 4 : 8 hours (inoculation, induction and titration)
Day 5 : 1 hour (observation & interpretation)

Fig: Schematic representation of the process of Bacterial

Transduction.

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Bacterial Transduction GeNeiTM Bacterial Transduction GeNeiTM

Materials Provided: Note:

The list below provides information about the materials • Read the entire procedure prior to starting the
supplied in the kit. The components should be stored as experiment.
suggested. • All microbiological operations should be done strictly
Quantity under aseptic conditions in Laminar Air Flow.
• Revive the strain as soon as the lyophilized vial is
Materials 6112300011730 Store
(5 Expts.)
opened
• Carry out the experiment within 2 weeks of reviving the
Donor strain 1 No. 4°C strain
Recipient strain 1 No. 4°C • Use sterile water for ampicillin & 70% ethanol for
Susceptible host 1 No. 4°C chloramphenicol preparation.
Phage lysate 50 µl 4°C • Ensure that no moisture is present on the surface of
hard agar plates
0.1M CaCl2 1 ml 4°C
• For preparation of media, refer appendix (Page 13).
Chloramphenicol 20 mg 4°C
Ampicillin 100 mg 4°C Procedure:
LB broth 100 g RT Day 1: Revival of Host
Agar 50 g RT 1. Break open the lyophilized vials of all three E.coli strains
1.5 ml vials 1 x 25 Nos. RT and re-suspend the sample in 0.1 ml of LB broth.
2. Streak 50 µl of Donor Strain on LB-C20 plate. Recipient
Note: Room Temperature (RT) not to exceed 20-25°C. strain on LB-A100 plate and Susceptible host on LB plate.
The other 50 µl into 5 ml LB broth with the respective
Materials Required: antibiotic.
Equipment : Centrifuge, Shaker incubators (set at 37ºC, 3. Incubate the plates and tubes at 30ºC overnight
30ºC and 42ºC), Water Bath (set at 60ºC). 4. Store the 5 ml culture tube at 4°C for inoculation on
Glassware : Test tubes, conical flasks, petri plates, day 3.
measuring cylinder, Centrifuge Tubes. Note:
Reagent : Distilled water, Ethanol. • LB-C20: LB with 20 µg/ml of Chloramphenicol.
Other Requirements : Micropipettes, 0.45 µm filters • LB-A100: LB with 100 µg/ml of Ampicillin.
(2 No)

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Bacterial Transduction GeNeiTM Bacterial Transduction GeNeiTM

Day 2: 6. Along with the above plate 50 µl of overnight Recipient

1. Inoculate 10 -15 colonies from the revived donor plate culture (Day 2 Step 10) and 50 µl of Donor culture (Day
(Day 1 Step 2) into 5 ml LB C20 and label as 'Donor 1 Step 4) on LB-C20, LB-A100 and LB-A100C20 plates.
tube'. 7. Incubate at 37ºC overnight.
2. Incubate at 30ºC in a shaker for 4 hours. 8. Observe the colonies in all plates. Proceed with positive
3. Add 10 µl of given Phage Lysate to the above 'Donor lysogen obtained on LB-A 100C 20 plate for further
Tube'. confirmation by induction.
4. Continue incubation at 30ºC for another 30 minutes 9. Store the plates at 4ºC.
5. Keep a 5 ml aliquot of sterile LB broth in a water bath, Day 4
set at 60ºC. Note: Inoculation for preparation of plating cells and
6. Add 2 ml of hot LB (which was maintained at 60ºC) to induction of the lysogens have to be started
the 'Donor tube' (Day 2 Step 4). simultaneously.
7. Mix well incubate at 42ºC for 20 minutes A) Preparation of Plating Cells
8. Shift the 'Donor Tube' to 37ºC and incubate for 3 hours 1. Inoculate 15-20 colonies from the revived Susceptible
9. Spin and take the supernatant. Filter it through host plate into 5 ml LB medium. (Revived on Day 1
0.45 µm filter and store at 4ºC for further use and label Step 2).
as 'Phage Lysate 2'. 2. Incubate for 4 hours at 37ºC.
10. Inoculate single colony from the revived Recipient strain 3. Spin down 1.5 ml culture in 3 different vials at
plate (Day 1 Step 2) into 5 ml LB-A 100; label as 6,000 rpm for 10 minutes at room temperature.
'Recipient Tube'. Resuspend each pellet in 100 µl of sterile fresh LB broth.
11. Incubate 'Recipient Tube' at 37ºC overnight Use these as 'Plating cells' for the titration.
(16-18 hours).
B) Induction of Lysogens
Day 3: 4. Keep a 5 ml aliquot of sterile LB broth in a water bath
1. Inoculate 100 µl from the overnight broth of Recipient set at 60ºC.
culture (Day 2 step 10) into fresh 5 ml LB broth with 5. To confirm the presence of phages in the lysogenized
100 µg/ml of Ampicillin. colonies, 10-15 colonies from LB-A100C20 plate (on which
2. Incubate at 37ºC for 3 hours. phage infected Recipient culture was plated) are
3. Take 50 µl of this culture in a test tube, add 50 µl of inoculated in to 5 ml LB-A100C20 (The plate from step
0.1M CaCl2 and 50 µl of Phage Lysate 2 (filtered 5 on Day 3) and label as 'Lysogenized culture tube'.
supernatant which was stored at 4°C, (Day 2, Step 9). 6. Incubate at 30ºC for 4 hours.
4. Incubate at 30ºC for 2 hours (static incubation). 7. Add 2 ml of hot LB (maintained at 60ºC as in Day 4
5. After 2 hours, plate 50 µl on LB-C 20, LB-A 100 and step 4) to the 'Lysogenized culture tube'.
LB-A100C20 plates (from Day 3 Step 4).
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Bacterial Transduction GeNeiTM Bacterial Transduction GeNeiTM

8. Incubate at 42ºC for 20 minutes. 17. Swirl to mix. Care must be taken to ensure that the
9. Then shift the tube to 37ºC and incubate for 3 hours. temperature of soft agar does not exceed 45ºC (as the
10. After 3 hours of incubation spin the culture, filter the host will die) or fall below 40ºC (as soft agar will solidify).
supernatant through 0.45 µm filter. Label as 18. Pour the mixture (phage-susceptible host cells with soft
'Concentrated lysate'. agar) immediately on LB hard agar plates. Label them
11. Dilute the supernatant from step 10 up to 10-3 dilution as concentrated, 10-1, 10-2 and 10-3 respectively and let
using LB as diluent. (Refer Fig). the agar solidify.
19. Repeat steps 13-15 for each dilution.
C) Phage Titration 20. Cover the plates and incubate overnight at 37ºC.
1 2 3
100 µl 100 µl 100 µl

900 µl LB Broth
Concentrated
Lysate 10-1 10-2 10-3
Fig: Diagrammatic representation of phage dilution
Note: Change the tip for each dilution
12. Take 100 µl of plating cells of Susceptible host in four
vials (plating cells obtained in step 3 Day 4).
13. Add 20 µl of the concentrated lysate (Day 4 Step 10)
and 20 µl of diluted (10-1 to 10-3) phage lysate to the
100 µl of the plating cells in each of the above vials.
14. Incubate all the 4 vials at 37ºC for 15 minutes.
15. Meanwhile melt the soft agar and maintain at 45°C
16. Pipette out the content of each vial into separate test
tube containing 5 ml of LB soft agar.

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Bacterial Transduction GeNeiTM Bacterial Transduction GeNeiTM

Observation: Appendix:
I. Screening of Transductants (Result of Day 3) Only Preparation of LB agar/broth (1 litre): Dissolve 25g of LB
Recipient strains which have acquired Chloramphenicol broth media in 800ml of distilled water. Adjust the pH to 7.0
gene from the Donor strain through the phage will grow with 5N NaOH (if necessary) and make up the volume to
on LB Ampicillin + Chloramphenicol plates. However the 1000 ml. Sterilize by autoclaving.
Recipient or the Donor alone will not grow on this
For LB agar, add 1.5% agar and autoclave.
combination of antibiotics.
II. Phage titration (Results of Day 5): Observe the plates for Preparation of soft agar: To prepare soft agar, add 0.8%of
plaques. Concentrated, 10-1, 10-3 plates will show distinct agar to LB broth. Boil to dissolve the agar. Aliquot 5 ml into
countable number of plaques. If the titre value is high test tubes and autoclave. Keep soft agar in molten state in a
countable number of plaques can be seen in 10-3 dilution water bath or oven, adjusted to 43-45ºC, just before use.
and complete chewing of the bacterial colonies will be
seen in the lower dilution. Preparation of Antibiotic solutions:
Chloramphenicol: Dissolve 20 mg of chloramphenicol in 1 ml
of 70% ethanol to get a stock concentration of 20 mg/ml.
Interpretation: Store at 4ºC. Use the antibiotic solution within 2 weeks.
I. This transduction experiment shows the transfer of
Chloramphenicol resistance gene from the Donor strain Ampicillin: Dissolve 100mg of given Ampicillin in 1ml of sterile
to the Recipient through a bacteriophage. water to get a stock concentration of 100 mg/ml Store at
4ºC. Use the antibiotic solution within 2 weeks.
As observed only the lysogenized/transduced recipient culture
is able to show resistance to Chloramphenicol (earlier which For antibiotic LB media: Add Chloramphenicol to LB broth or
was sensitive to Chloramphenicol agar at a final concentration of 20 µg/ml, when the
temperature of the medium is around 40-45ºC. Similarly add
Record the results as follows:
Ampicillin to LB broth or agar at a final concentration of
LB LB Ampicillin LB Ampicillin & 100 µg/ml, when the temperature of the medium is around
Chloramphenicol Chloramphenicol 40-45ºC.
Donor strain + - -
Recipient strain - + -
Transduced/
Lysogenized + + +
strain

Where + indicates growth

- No growth
II. Phage titration results confirm the presence of phage
particles in the lysogenized recipient strain
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Bacterial Transduction GeNeiTM Bacterial Transduction GeNeiTM

Ordering Information
Product Size Cat #

GeNei™ Bacterial 1 EA 6112300011730

Transduction Teaching Kit
(Consumables for 5 experiments)

Email:
Sales: sales@geneilabs.com

Customer Support: techsupport@geneilabs.com

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