Team:Wageningen UR/Experiments

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Wageningen UR iGEM 2016

 

General Protocols

Mite Shower

This video shows how we checked our beehive frames for the presence of the queen before we swapped the frames.

Varroa Isolates

Identifying Isolates from Varroa destructor

One of the methods we used to find our own Bacillus thuringiens isolate was based on a combination of the procedure used by Rampersad et al.1. and Alquisira-Ramírez et al.2. It combines the genus’s ability to form spores with a Coomassie stain that allows for visualization of Cry toxins and protein-rich parasporal bodies.

The most important aspect of this protocol is a source of fresh Varroa mites. The longer they have been dead, the more dessicated they will be. This will complicate the sterilisation step. The easiest way to handle the dead mites is with a fine , wetted brush. This will prevent damage to the mites.

To promote sporulation of the isolates, sporulation plates were prepared. These are regular LB agar plates with the following salts added:

  • 0.14 mM CaCl2
  • 0.20 mM MgCl2
  • 0.01 mM MnCl2

Instructions

  • Transfer 10 mites to a microcentrifuge tube
  • Sterilize with 1 mL of a 2.5% bleach solution – only immerse for 5 seconds
  • Transfer the mites to a sterile microcentrifuge tube
  • Wash with 1 mL of sterile tap water
  • Replace the water with 500 µL LB
  • Macerate the mites in the LB with a pipette tip or a glass rod
  • Heat the tubes in a heatblock for 15 minutes at 65°C
  • Take 200 µL of LB and plate on sporulation plates
  • Spin down the remaining LB, pour off and resuspend – plate the resuspension

The plates should be incubated at 30°C for at least 2 days, preferably longer. Any appearing colonies with Bacillus cereus-like colony morphology (round, white colonies) should be streaked on sporulation plates and grown for at least 2 days.

Not all white, round colonies with the ability to form spores will have rod-shaped cells. The following stain stains vegetative cells, spore cell walls and protein-rich bodies such as Cry toxins and parasporal inclusions.

  • 50% methanol
  • 40% demineralized water
  • 10% acetic acid
  • 0.1g Coomassie Brilliant Blue R per 10 mL staining solution

The staining solution and the destaining solution are the same except for the addition of Coomassie Brilliant Blue R.

To stain the isolates, the following instructions should be followed. Remember to work in a fumehood as the staining and destaining solutions are both toxic.

  • Scrape a small amount of colony with an inoculation loop
  • Mix colony with a drop of water on a microscopy slide
  • Let the slides air dry, then heat fix by passing over a flame 3 times
  • Immerse the slides in staining solution for 45 seconds
  • Immerse the slides in destaining solution for 60 seconds, agitate them for the first 10 seconds
  • Pour off the solution; let air dry
  • If the slides are too wet, gently place them upside down on tissue paper to dry
  • The slides are now ready for brightfield microscopy!

When imaging the colonies, look for colonies with large, rod-shaped cells. If you look carefully, you may be able to see parasporal inclusion bodies or Cry toxins. It is a good idea to get a control strain that produces Cry toxins to have a clear idea of what you are looking for.

Protein Extraction and SDS-PAGE

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16s rRNA PCR

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LC-MS/MS

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Protein Engineering

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In Vitro Assay

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References

      1. Rampersad, J., & Ammons, D. (2005). A Bacillus thuringiensis isolation method utilizing a novel stain, low selection and high throughput produced atypical results. BMC microbiology, 5(1), 1. ↩

    2. Neethu, K. Alquisira-Ramírez, E. V., Paredes-Gonzalez, J. R., Hernández-Velázquez, V. M., Ramírez-Trujillo, J. A., & Peña-Chora, G. (2014). In vitro susceptibility of varroa destructor and Apis mellifera to native strains of Bacillus thuringiensis. Apidologie, 45(6), 707-718. ↩