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Conjugation.md

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Triparental conjugation and streaking the bacteria on plate

Sofie Thijs (sofie.thijs@uhasselt.be)

date: 23/10/2020

   

On-campus

Perform the conjugation

Follow the instructions on page 16 in the lab manual, to perform the triparental conjugation.
The assistants will demonstrate how you work sterile.

Lunch break

Choose the selective growth medium

Choose your selective medium to be able to select your transconjugant. Questions to help you:

  • Which nutrient medium are you going to use? Hint: you want only your receptor+gfp-vector strain (= transconjugant) to grow, not the donor and helper E.coli
  • Are you going to add certain supplements to your medium as carbon or nitrogen source?
  • Are you going to incubate at a certain temperature?
  • Are there other things you are going to do, other ways you know of to select your transconjugant?

Plate the bacteria on your selective medium

Follow the instructions on page 17 in the lab manual, to streak your bacteria on plate after conjugation.
The assistants will also show you how to do this.

   

Online

Perform the conjugation

Follow the instructions on page 16 in the lab manual, to know how to perform the triparental conjugation.
You are going to add all 3 bacteria on top of each other, the bacteria need to make contact, in order for conjugation to happen.

Lunch break

Choose your selective growth medium

Choose your selective medium to be able to select your transconjugant. Questions to help you:

  • Which nutrient medium are you going to use? Hint: you want only your receptor+gfp-vector strain (= transconjugant) to grow, not the donor and helper E.coli
  • Are you going to add certain supplements to your medium as carbon or nitrogen source?
  • Are you going to incubate at a certain temperature?
  • Are there other things you are going to do, other ways you know of to select your transconjugant?

Mail me the medium for feedback (sofie.thijs@uhasselt.be).

Plate the bacteria on your selective medium

When you have confirmation of the correct medium, you can now streak the bacteria after conjugation, on your selective medium. Watch the instruction video how to do streak plate method, and then do it yourself using this virtual lab module.

Your bacteria will grow over the weekend! Let's check the results on monday morning together.

References

  • If you have time left: check this video about conjugation. Use this to discuss your findings of the conjugation results.
  • More details about how the pilus is formed.
  • If you want to refresh both conjugation and transduction