Reference no: EM133111116

MI2011 Microbial Diversity - James Cook University

Practical: Basic Molecular Biology of Bacteria - Mechanisms of Gene Transfer

In this practical exercise, you will attempt the conjugative transfer of violacein genes (which confer a violet pigment to the host cell) and kanamycin resistance. You will be provided with two strains of E. coli, one of which will be the donor and the other the recipient. Based on colony morphology and growth of the species before and after conjugation, you should be able to determine which is the donor and which is the recipient and what order the donated genes sit on the conjugative plasmid.

You will be supplied with:
1 x LB agar plate of E. coli Q358/R751 (24 hour culture) 1 x LB agar plate of E. coli 1B609 (24 hour culture)
LB agar plates containing kanamycin (4) LB agar plates containing streptomycin (4)
LB agar plates containing kanamycin and streptomycin (4) LB broth
Microfuge tubes

A flow chart is provided after the instructions to aid in following the instructions.

1. Note the morphology of each E. coli strain, with particular regard to any colour pigments, size and smoothness/roughness of the colonies. Use the space below to make these observations.

2. Add 500µl of LB broth to a microfuge tube. Using a sterile loop, suspend a 0.5 McFarland suspension (only just turbid) of E. coli IB609 in this 500µl LB. Flick/shake/agitate tube to break any clumps.
a) Prepare a suspension by obtaining a fresh, pure culture of the organism and inoculating a suitable broth.
b) Visually compare the turbidity of the created suspension with that of the McFarland standard demonstration provided.
c) If the created suspension is too light, inoculate with additional organisms until turbidity matches that of the standard. If dilution is necessary, use a sterile pipette and add sufficient broth to obtain a turbidity that matches that of the standard.

3. Repeat step 2 with E. coli Q358/R751 in a separate microfuge tube.

4. Streak both E. coli cultures onto each of the LB agar plates containing the single antibiotics and spread plate onto the LB agar plates containing both antibiotics - as per step 9 below (you should now have inoculated 6 plates):
a) Kanamycin (LB kan) (streak plate)
b) Streptomycin (LB strep) (streak plate)
c) Kanamycin & Streptomycin (LB k&s) (spread plate)

5. Set these plates aside for later incubation.

6. Aseptically transfer 1 culture into the other, label top of tube clearly with your name and mix by tube inversion.

7. Incubate your E. coli mixture in the rack provided in the 37°C hotblock for the allotted time assigned to you by the instructors (10 or 30 min).

8. Streak the mixture on each of the single antibiotic LB agar plates (LBkan, LBstrep).

9. Using the spread plate method, pipette 100µl of the mixture onto the mixed
antibiotic (LBk&s) plates. Details of this method are below;
a) Add the bacteria to the plate.
b) Remove the lid from the ethanol container and dip the glass spreader in.
c) Replace the lid onto the ethanol container.
d) Rapidly pass the glass spreader through the Bunsen burner once and then hold the glass spreader in the zone of sterility to allow the alcohol to burn off. Prolonged exposure to heat will cause the glass rod to become deformed or shatter.
e) Allow the glass spreader to cool and then spread the sample evenly over the entire surface of the agar plate by moving the spreader backward and forward across the plate while turning the plate with your other hand. Do not lift the plate from the table during this process.
f) Re-sterilise the glass spreader.
g) Replace the lid onto the agar plate and allow the sample to dry onto the agar before inverting the plate for incubation.

10. Return the liquid bacterial mixture to the incubator as soon as possible to incubate overnight.

11. Place a rubber band around all inoculated agar plates and place in the box for incubation at 37oC for 48 hours

Question 1: Define the terms donor strain and recipient strain and Identify which of the cultures you used were the donor and recipient strains, using justifications and reasoning based on expected results (provided on LJCU). Did conjugation occur in your case (explain why/why not)?

Question 2: Describe the process by which the vio gene was transferred from the donor to the recipient.

Question 3: Explain why you streaked both cultures onto three antibiotic-containing agars at the start of the experiment. Explain how both antibiotics being added together to LB agar will select for transconjugants.

Question 4: Explain why you think you were asked to spread your mixture after two different time periods? How could this timed procedure (if you did more time intervals) help you in the study of the plasmids?

Question 5: From the literature, do you consider conjugation to be the primary method for transfer of antibiotic resistance between bacteria in nature? If not, which method do you consider to be more likely, use scientific literature to justify your choice?

Attachment:- Microbial Diversity.rar