General Protocols

Mite Shower

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

Protein Engineering

YOUR TEXT HERE

In Vitro Assay

YOUR TEXT HERE

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.This protocol was used for these results.

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.

In Vivo toxicity

The toxicity assay to determine in vivo toxicity was based on the assay performed by Alquisira-Ramírez et al. ² Protein extracts were diluted to 100 µg/mL in a 0.1% Tween-80 Tris-HCL buffer (pH 7.5). Per sample, 5 mites were dipped in the protein solution for 5 seconds, then sieved with sterile filter paper. They were then put on an Apis mellifera pupa and mortality was observed every 24 hours. As a negative control, Tween-80 buffer without protein extract was used as well as Bacillus subtilis protein extracts. As a positive control, Tenebrio molitor larvae were used in combination with protein extracts from Bacillus thuringiensis tenebrionis.

16s rRNA PCR

16s rRNA PCR can be used to identify bacterial isolates. The 16s rRNA region consists of highly conserved as well as variable regions; primers anneal to the conserved regions, so the variable regions can be sequenced and used to identify an isolate to the genus level.

The following primers were used to amplify the 16s rRNA region:

27f: 5’ AGRGTTTGATCMTGGCTAG
1492r: 5’ TACGGYTACCTTGTTAYGACTT

OneTaq was used for the PCR reaction.

Ingredient	Volume (µL)
dH₂O	36.75
5x OneTaq buffer	10
dNTP mix (10 mM)	1
27f primer (10 µM)	1
1492r primer (10 µM)	1
OneTaq polymerase	0.25

A sterile pipette tip was used to transfer some cells from the single colonies to the PCR tubes. This was done in a UV cabinet with UV-sterilized gloves and tools to prevent contamination. Water was used as a negative control, Escherichia coli as a positive control. The following program was used with 35 cycles:

Step	Temperature in °C	Time
Predenaturation	95	10 minutes
Denaturation	95	40 seconds
Annealing	55	40 seconds
Extension	68	1 minute
Final Extension	68	5 minutes

The PCR products were run on a 1% TAE gel for 30 minutes at 100V to verify the length. After PCR clean-up, samples were sent to GATC for LightRun sequencing.

LC-MS/MS

LC-MS/MS was performed on bands containing a 100 kDa protein. For the LC-MS/MS, the following protocol was followed¹.

Prepare an SDS-PAGE gel with the protein of interest
When handling the columns and peptides, use wetted nitrile gloves to prevent keratin contamination
Cut out bands at the height of the protein, as well as some distance above it
Treat the gel bands with 10 mM DTT at pH 8 for 1 hour at 60°C
Treat with 20 mM iodoacetamide at pH8 for 1 hour at room temperature
Cut bands into 1 mM² pieces
Digest for 2 hours at 45°C with 5 ng/µL trypsin solution
Extract peptides by elution in a 10% trifluoro-acetic acid solution
Clean peptides with a C18 µColumn

Prepare the column as follows:
Cut 1.6 mM disk from a C18 Empore disk
Transfer the disk to a 200 µL pipette tip
Pipet 200 µL methanol on the disk
Pipet a 50% slurry of Lichroprep C18 column material into the methanol
Wash once with 100 µL methanol
Equilibrate with 100 µL 1 mL/L formic acid in water
Prevent the column from running dry or being obstructed by air bubbles

Dissolve samples in 100 µL 1 mL/L formic acid and run through a column
Wash the columns once more with formic acid
Elute samples in 50 µL 50% acetonitrile and 50% 1 mL/L formic acid into a low-binding microcentrifuge tube
Concentrate samples with a vacuum concentrator to remove acetonitrile
Your samples are ready for LC-MS/MS!