Following gene transfer by conjugation in bacteria
This practical follows the horizontal transfer of genes from one bacterial strain to another by conjugation. This is one of three ways in which bacteria can pick up genes within the lifetime of individual cells. Horizontal transfer produces an immediate change in the genetic information available in a cell and therefore can result in an almost immediate change in the characteristics of a bacterial cell – for example, conferring resistance to antibiotics. As a process of change, or evolution, it acts more quickly to produce a change to a whole population than mutation followed by selection where genes are transferred only vertically to offspring.
The donor strain of Escherichia coli bacteria (E. coli HT-99) carries a plasmid with a gene that gives resistance to the antibiotic chloramphenicol. The recipient strain of bacteria (E. coli J-53R) carries on its chromosome a gene that gives resistance to the antibiotic rifampicin. Mix liquid cultures of the two strains of bacteria and allow them to ‘mate’. Plate out samples of donor, recipient and ‘mated’ cells on three kinds of selective media – one containing rifampicin, one containing chloramphenicol and one containing both antibiotics.
The procedure is adapted from Welcome to the X-Bacteria in the Survival Rivals kits produced by the Wellcome Trust as part of the Darwin200 initiative. Go to www.survivalrivals.org for more details. The materials are also available – separately or in the form of kits – from other suppliers. See Web links at the bottom of this page.
Students’ skills in aseptic technique will be improved if you can demonstrate the methods to them before they carry out each stage of the practical.
The procedure involves living microbiological material and processes taking a fixed amount of time. It is essential to prepare the cultures a suitable amount of time in advance. Carrying out all the stages of the practical takes three teaching sessions. Some stages must take place within fixed times, so this needs careful planning. It is possible for different teaching groups to carry out different steps of the procedure and for all to review the results. This might make it easier to fit into your timetable and limit the consumption of materials.
Apparatus and Chemicals
For each group of students:
Access to a water bath, controlled at 50 °C
Access to hand-washing facilities with soap and hot water
Waste containers holding Virkon solution for disposal of Pasteur pipettes
Paper towels (to swab down benches)
Nutrient broth, sterile, 10 cm3
Overnight culture of E. coli J53-R, 10 cm3
Overnight culture of E. coli HT-99, 10 cm3
Sterile syringe, 1 cm3, 2
Adhesive tape (to secure the lids of Petri dishes)
Marker pens (to label plates)
For the class – set up by technician/ teacher:
An autoclave or pressure cooker (to prepare and dispose of microbiological materials)
An incubator, at 30 °C
Conical flasks, 500 cm3, 3 (to prepare growth media)
Autoclavable disposal bags, at least 4 (for contaminated paper towels, cultures, plates etc)
Active bacterial cultures, 10 cm3 of each for each working group (Note 5)
Nutrient broth, sterile, 10 cm3 for each working group
Antibiotic solutions in methanol (Note 6)
Nutrient agar plates with added antibiotics, one of each type for each working group (Note 7)
Health & Safety and Technical notes
Carry out a full risk assessment before planning any work in microbiology (see Note 1 for more details). This should include consideration of the knowledge and skills of teachers and technicians as well as the competence of students. It is essential that all cultures and solutions containing microorganisms and antibiotics are disposed of appropriately (Note 2).
Check the standard techniques for more details of Aseptic techniques, Making up nutrient agars, Pouring an agar plate, Maintaining and preparing cultures and Incubating and viewing plates.
2 Plan how you will dispose of the material contaminated by this procedure before you begin. Used cultures and platesmust be sterilised by autoclaving before disposal – or disposed of by incineration. Paper towels and disposable equipment should also be autoclaved before disposal. This is especially important as the bacteria used are resistant to antibiotics and some now have multiple resistances.
3 Incubate at 30 °C. This is the preferred temperature for incubating these organisms for this practical. Incubating below human body temperature reduces the risk of cultivating pathogens that could affect people. Between stages 2 and 3 you can store the plates in a fridge after initial incubation, if there is a longer gap than 24 hours between lessons. Bring to room temperature slowly to reduce condensation that would make it hard to see the bacterial growth. If the lids have a film of moisture, replace them with fresh, dry lids.
4 Lab coats should be worn for this practical and laundered before subsequent use. If any clothing is severely contaminated, soak in disinfectant before laundering. Gloves should be worn by any students who have cuts or abrasions on their skin that cannot easily be covered with plasters. There is no need for disposable gloves – gloves reduce manual dexterity for some students.
5 To make sure your cultures are growing actively, streak onto agar plates and then inoculate broth from the plated culture. See Maintaining and preparing cultures of bacteria and yeasts. All microorganisms should be regarded as potentially harmful. However, the strains of E. coli used present minimum risk in the context of good laboratory practice.
6 Antibiotics in aqueous solution are damaged or broken down by heat, so you will usually have them supplied as powders. If you cannot safely weigh out very small quantities, you need to dissolve the full contents of any vial provided (which will be of a known mass) in 5 cm3 of methanol first – see below. Then dilute into sterile distilled water at a final volume of 100 cm3 before adding aseptically to the nutrient agar. The CLEAPSS Hazcard describes methanol as HIGHLY FLAMMABLE, and TOXIC by inhalation, in contact with skin, and if swallowed.
Once dissolved in methanol (Note 7), wrap the bottles of antibiotic in aluminium foil; then they can be stored in the fridge for up to a week. Safe handling of antibiotics means avoiding inhalation and skin contact – by working in a fume cupboard, wearing gloves and eye protection, as well as by tapping the vials gently to knock the powder to the bottom before opening. Dispose of any excess antibiotic solution by autoclaving to denature it, before pouring it down a sink with plenty of tap water.
7 Making up antibiotic plates: Allow approximately 20 cm3 of medium per plate. For 12 working groups, this is approximately 250 cm3 of each kind of agar. You are aiming for final concentrations of:
• chloramphenicol at 25 µg per cm3, that is 25 x 250 = 6250 µg = 6.25 mg chloramphenicol in a final volume of 250 cm3
• rifampicin at 100 µg per cm3, that is 100 x 250 = 25000 µg = 25 mg rifampicin in a final volume of 250 cm3.
Each working group needs three plates – all containing nutrient agar, but each with a different combination of antibiotics. Make up three batches of nutrient agar by adding the correct amount of solutes for a final volume of 250 cm3 to distilled water in conical flasks. (See Making up nutrient agars.) Flasks A and B should contain 225 cm3 of water as you will be adding one measure (25 cm3) of antibiotic to each. Flask C should contain 200 cm3 of water as you will be adding two measures of antibiotic to it. Maintain the nutrient agar at 50 °C in a water bath until you pour the plates. Label the plates before pouring so it is clear to students which antibiotics are present.
There are no ethical issues associated with this procedure, as the bacteria with antibiotic resistance will be destroyed at the end of the practical and not released into the environment.
Take care when handling antibiotic powders to avoid inhalation of the powder and to avoid contact with the skin. Staff with known sensitivities should avoid contact with the antibiotics.
Take care working with methanol to dilute the antibiotics as it is highly flammable and toxic by inhalation, ingestion and by skin contact.
Collect all contaminated material to make it easy to clean or dispose of: use one autoclave bag for reusable glassware, another autoclave bag for disposable materials and a discard jar of Virkon for disposable loops and waste liquid. Dispose of each item appropriately at the end of the practical.
a Make streak cultures from your supplied bacterial slope cultures a few days before running the practical. Make up cultures in broth from these streak plates at least 2 days before use.
b Prepare plates of nutrient agar with added antibiotics the day before use.
Investigation Stage 1
c Wash your hands with soap and water.
d Disinfect the bench surface with Virkon. Wait 10 minutes and then dry it off. Light a Bunsen burner near where you are working to create an upward flow of warm air. This will carry away microorganisms that could contaminate your cultures.
e Label the bottle of sterile nutrient broth ‘mating’.
f Use a sterile syringe to transfer 2 x 0.9 cm3 (a total of 1.8 cm3) of the culture of E. coli J-53R aseptically into your bottle labelled ‘mating’.
g Discard the used syringe into a waste container of disinfectant.
h Use a new syringe to transfer 0.2 cm3 of the culture of E. coli HT-99 aseptically to your bottle labelled ‘mating’.
i Discard the used syringe into a waste container of disinfectant.
j Retain the two E. coli cultures and label all three bottles with your initials.
k Place the three cultures in an incubator at 30 °C for 4-16 hours.
l Disinfect the work area again.
m Wash your hands again with soap and water.
Investigation Stage 2
n Wash your hands with soap and water.
o Disinfect the bench surface as before.
p Light a Bunsen burner in your work space to maintain aseptic conditions and to flame your wire loop when necessary.
q Collect three nutrient agar plates – one containing rifampicin only, one containing chloramphenicol only and one containing both antibiotics. Collect your bacterial cultures from last lesson.
r Turn over each plate so you can write on the bottom, not the lid. Use the marker pen to mark three segments on each plate as shown in the diagram. Label the segments ‘Donor’ (this will be the HT-99 strain), ‘Recipient’ (this will be the J-53R strain) and ‘Mating’ (this will be the mixture of the two strains from last lesson). Make sure you check which antibiotics are in which plate and label them if it is not clear (in small characters at the edge of the plate).
s Arrange the plates on the bench in front of you.
t Sterilise a wire loop in a Bunsen flame, and use the sterile loop to streak the ‘Donor’ section of each plate with your Donor culture (HT-99). Sterilise your loop in the Bunsen flame again.
u Repeat stage t with a freshly sterilised loop and streak the ‘Recipient’ section of each plate with your Recipient culture (J-53R). Dispose of your loop into the disinfectant solution.
v Repeat with a third loop and your 'Mating' culture. Dispose of the loop into disinfectant solution again.
w When the agar has absorbed any excess liquid, tape the lids onto the plates (but do not seal), invert the plates and incubate them for 24 hours at 30 °C. (See Standard technique: Incubating and viewing plates safely for more information.)
x Wipe down the bench with disinfectant.
y Wash your hands with soap and water.
Investigation stage 3
z Inspect the plates and record results to show which bacteria have grown successfully on which plate. Do not open the plates and dispose of them into the autoclave bags provided.
There are three principal ways in which genes can be transferred horizontally in bacteria:
- transformation in which naked DNA is picked up by cells. Many bacteria are naturally able to do this (‘competent’) and it is probably the most common mechanism of horizontal transfer in nature, but the DNA Involved is often degraded by enzymes or performs no function.
- transduction in which the transfer is mediated by a virus. This is an important gene transfer mechanism in the generation of genomic diversity and bacterial evolution.
- conjugation in which DNA is transferred between cells via a special tube. Transfer is one-way (from donor to recipient). The donor is referred to as ‘male’, the recipient as ‘female’ and the conjugation tube as a ‘sex pilus’ or tube. In this case, the donor contains a plasmid – a small ring of DNA that can replicate independently of the main bacterial chromosome. When it replicates, a single strand of plasmid DNA is formed which passes through the pilus. The complementary strand is synthesised in the donor cell and a new double-stranded, circular plasmid forms. Plasmids typically carry a few non-essential genes, and often the genes that are responsible for resistance to antibiotics.
Recent media reports of food poisoning due to E.coli may make students nervous about the risks of handling this microorganism. It is important to explain that there are several strains of E.coli and the strains used here (E.coli HT-99 and E.coli J-53R) are harmless. E.coli VT-157 is a virulent strain that causes food poisoning.
Students could carry out research into the antibiotics used, and the details of conjugation or other methods of horizontal gene transfer as part of homework activities associated with this practical.
There is scope to develop in-depth discussions of the emergence of antibiotic resistance in bacterial populations, the social and healthcare consequences of antibiotic resistance, and the special features of microbial evolution that result from this mechanism of gene transfer.
There is an animation of conjugation on the Survivalrivals website at www.survivalrivals.org/the-x-bacteria/animation.
Health & Safety checked, September 2009
Download the student sheet Following gene transfer by conjugation in bacteria (82 KB) with questions and answers.
A kit of equipment to run this project is available for purchase from Philip Harris. The kit includes all consumable materials to carry out conjugation between two strains of E. coli carrying different genes for antibiotic resistance (one for rifampicin and one for chloramphenicol).
Edvotek offers several kits for transformation practicals, including one following antibiotic resistance – Transformation of E. coli with Plasmid pBR322.
NCBE also offers a kit – the Bacterial transformation kit – of materials and consumables needed to run a transformation practical. NCBE’s literature is available to download from the site at www.ncbe.reading.ac.uk/NCBE/MATERIALS/DNA/transformation.html
(Websites accessed October 2011)