Varroa Isolates

Identifying Isolates from Varroa destructor

One of the methods we used to find our own Bacillus thuringiensis isolate was based on a combination of the procedure used by Rampersad et al.⁴. and Alquisira-Ramírez et al.⁵. 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 al⁵. 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, T. molitor larvae were used in combination with protein extracts from B. 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

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:

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 followed⁶.

• 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!

Cas9 kill switch

Mutagenesis PCR

To make mutations in dCas9, mutagenesis PCR was used according to the Quikchange protocol. The adapted protocol that worked best for us is shown here.
Primers with mismatches to the template on the site of the mutation were designed using their online tool. The following PCR mixture was made:

Most of the time, 100 ng template worked best for us. Additionally, 5 µL of 5x GC enhancer was needed to run the Ala10 → TAG PCR reaction successfully.

Then, the following cycling program was used to amplify the DNA:

Step 2-4 were repeated 30x.

PCR products were checked by gel electrophoresis. If amplification was successful, PCR products were cleaned using the Zymo PCR cleanup kit according to the accompanying protocol and eluted in water. The cleaned samples were digested for I.5 hours with DpnI, cleaned again, and 5 µL of the eluted sample (in water again) was used to transform electrocompetent cells. Resulting colonies were miniprepped and verified by sequencing.

Yeast assembly

To construct pEVOL-Asp, a construct containing an aminoacyl synthetase (aaRS) and tRNA_CUA (ref) suitable for incorporating Aspartate in response to the TAG stopcodon, yeast assembly was used. In this method, linear DNA fragments are transformed into Saccharomyces cerevisiae. To select for positive clones, one of the fragments is a part of the yeast expression vector pYES2. pYES2 contains the URA3 gene, which releases auxotrophy for uracil, as well as a 2µ ori for high copy maintenance in yeast. The other fragments included two IDT gBlocks containing the aaRS and tRNA_CUA, as well as part of pEVOL-pAzF⁷ containing the arabinose promoter and CAT resistance gene.

Primers to generate fragments for yeast assembly were made using the NEBuilder for Gibson assembly, with 40 bp overlap between fragments. PCR was performed as usual (see the protocol), fragments were checked by gel electrophoresis and PCR products were purified using the PCR cleanup kit from Zymo.

Homemade heat-shock competent S. cerevisiae cells were incubated at RT for 10 minutes prior to mixing with ~400 ng of each PCR product and 100 µg sheared salmon sperm DNA (Sigma Aldrich). 700 µL of 1x LiAc/ 40% PEG-3350/ 1x TE was mixed in and solutions were incubated at 30°C for 30 minutes. Next, 88 µL DMSO was added, samples were mixed and heat shocked at 42°C for 7 minutes. Cells were pelleted in a microcentrifuge, washed with 1 ml 1x TE, resuspended in 100 µL 1x TE and plated on YPD plates. Plates were incubated over the weekend at 30°C.

To isolate the DNA from possible correct clones, a modified miniprep protocol was followed.
Colonies were inoculated in 1.5 ml YPD medium and grown overnight while shaking at 30°C. Cells were harvested in a microcentrifuge (13000 rpm, 5 minutes) and the pellet was resuspended in 100 µL STET buffer. To lyse the cells, they were disrupted in a bead beater, another 100 µL STET was added and samples were boiled for 3 minutes in a heat block. Samples were spinned down again in a microcentrifuge, and the supernatants were used to proceed with miniprep as usual (hyperlink). This gave fairly low plasmid yields (~30 ng/ µL), but enough to do PCR and transform into E. coli.

Correct construction of the plasmid was confirmed by PCR and sequencing.

YNB-UDM medium/plates

10x TE

pH was adjusted to 7.5.

10x LiAc

pH was adjusted to 7.5 and filter sterilized.

STET buffer

Cloning of gRNA plasmids using annealed oligos

Cloning was performed in the same way as normal digestion-ligation cloning, but annealed oligonucleotides were used as insert.

Oligos were annealed as in Jao et al⁸, but using 1x ligase buffer instead of NEBuffer 3.
Two complementary oligos corresponding to the target sequence were diluted to 10 µM each in T4 ligase buffer (NEB) to a final volume of 20 µL. To anneal them, the following protocol was used:

5 min denaturation at 95ºC, cooling to 50ºC at -0.1ºC per second, remaining at 50ºC for 10 minutes, followed by cooling to 4ºC at -1ºC per second.

The annealed oligos were cloned into pT7-gRNA (Addgene plasmid # 46759). pT7-gRNA was digested with BsmBI and BglII for 1 hour. Cut plasmids were checked by gel electrophoresis, and digested bands were purified from the gel (hyperlink).

Next, we proceeded with ligation as usual, diluting the annealed oligos 16x to add 1 µL to 25 ng digested pT7-gRNA. Ligation mixtures were diluted 2x, and 1 µL was used for electroporation.

in vitro RNA transcription and purification

Note: always use gloves and filtered tips when working with RNA, as RNAses can contaminate your samples

in vitro transcription

Prior to transcriptions, pT7-gRNA plasmids were digested with BamHI and purified. RNA guides were transcribed in vitro using the HiScribe high yield RNA synthesis kit from NEB. Reactions were assembled using the following protocol:

Mixtures were incubated at 37°C for 4 hours and digested with 2 µL DNAseI for 15 minutes at 37°C.

Checking transcription products on urea gel

In vitro 5 µL of the transcription products, as well as 1 µL low range ssRNA ladder, were mixed with 2x RNA loading dye, incubated at 90°C for 5 minutes and put on ice immediately thereafter.
1.5mm urea gels were poured and left to polymerize for >30 minutes. Gels were assembled and prerun for 15 minutes (15 mA for 1 gel, 35 mA for two gels). As a running buffer, 0.5x TBE was used. Before loading the transcription products, wells were flushed with running buffer to remove urea from the wells.
Samples were loaded and run for ~1 hour.

To visualize the RNA on the gels, they were stained with SybrGold (Thermo Fischer Scientific) in 100 mL 0.5x TBE for ~20 minutes. After staining, bands on the gel can be visualized with UV light. The RNA transcripts might appear as big, uncolored blobs; in that case, there is so much RNA that the dye does not penetrate the gel completely.

If gels showed RNA bands of the right size, the remainder of the transcription products was loaded on the gel for subsequent purification.

RNA purification from acrylamide gel

RNA bands of the correct size were cut from the gel, cut in small pieces with a syringe and incubated overnight in 1 ml RNA elution buffer.
The next day, the elution buffer was divided over 3 eppies and 990 µL of pre-chilled (-20°C) 100% EtOH was added. Samples were incubated for 1 hour at -20°C, spun down in a microcentrifuge for 20 minutes, 13000 rpm, at RT. At this point, a pellet can be seen that is usually translucent, but in our case whitish and flakey. Pellets were washed with 1 ml 70% EtOH (combining them again), spun down again for 5 minutes, 13000. The majority of EtOH was removed, and the remainder was evaporated at 55°C in a heatblock. Pellets were resuspended in 20 µL MQ, after which purity and concentration was measured with Nanodrop.
5x TBE buffer

pH should be ~8.3.

7M urea 10% PAGE gel (for two pieces, 1.5 mm)

This solution was filter sterilized, 0.45 µm. 0.125 mL 10% APS and 0.025 mL TEMED were added subsequently, after which polymerization starts.

2X RNA loading dye

RNA elution buffer

pH was adjusted to ~7. This needs to be done very carefully by the drop. If a precipitate is visible, incubate at 37°C before measuring pH.

Cas9 expression and FPLC purification

Day 1
Cas9 was expressed in a recoded E. coli strain lacking TAG stopcodons and release factor 1(C321dA; ref), but higher expression can probably be achieved using a bacterial strain more suited to protein expression.

10 ml overnight cultures were prepared of C321dA transformed with Cas9-expresso, dCas9-expresso, Ala10TAG-expresso + pEVOL, or Ala840TAG-expresso + pEVOL. Cultures were grown in LB in the presence of 35 µg/mL kanamycin (expresso constructs) and 25 µg/mL chloramphenicol (pEVOL constructs) when appropriate.

Day 2
Overnight cultures were diluted 100x in 0.5 L LB, with appropriate antibiotics, and divided over two 1L erlenmeyers. Cultures were were grown to an OD600 of about 0.8 at 37ºC while shaking. Next, cultures were put at 20ºC shaking for 30 minutes (cold shock) to induce expression of chaperones that aid in protein stability.
Cas9 expression was induced by adding rhamnose to 0.2%. In cultures that contained a pEVOL vector, expression of the aminoacyl synthetase was induced by adding arabinose to 0.2%. When appropriate, at this time also the synthetic amino acid was added to a concentration ranging from 200 µM to 1 mM. Cultures were shaken overnight at 20ºC.

Day 3
All centrifugation steps were performed at 4 ºC, and samples were kept on ice.
Cells were harvested by centrifugation at 4000 x g for 15 minutes. Supernatant was removed, and cells were washed once in 50 ml Tris-HCL buffer, pH7. After spinning down the cells again, they were resuspended in 10 ml cold His buffer A. Cells were lysed using a chilled French press cell (pressure ~30,000 psi) and lysed samples were spinned at 16000 x g for 10 minutes. Supernatants were collected, filtered (0.45 µm filter) and purified using FPLC.

Since C321dA is not optimized for protein expression, we aimed to reduce protein breakdown by adding cOmplete protease inhibitor to our samples before lysis. However, high yield of Cas9 was also observed when this protease inhibitor was omitted.

Using the expresso vector for Cas9 expression introduced a C-terminal his-tag to our Cas9 constructs, which was used for protein purification. An AKTA Purifier system was used, with a 1 mL HisTrap HP column (GE Healthcare Life Sciences) that was equilibrated with His buffer A . The cell-free extracts were loaded on the column, and the column was washed by addition of 2% His buffer B to already remove some unspecifically bound proteins. Elution was performed with a gradient of His buffer B.

His buffer A

pH was adjusted to 8.0.

His buffer B

pH was adjusted to 8.0.

in vitro Cas9 cleavage assay

Cleavage assays were performed according to the NEB protocol, but with our own Cas9 samples and homemade 5x Cas9 assay buffer.

The following reaction mixtures were assembled:

This mixture was allowed to incubate for 10 minutes at 25°C, followed by addition of 3nM (final concentration) substrate DNA (in our case, 200 ng of a 4100 bp PCR product). The, it was incubated for 1-1.5 hours at 37°C.

Cleavage was assessed by gel electrophoresis on agarose gels, but stained after running with SybrGold (Thermo Fisher Scientific) for ~25 minutes instead of SybrSafe.

5x Cas9 assay buffer

pH was adjusted to 6.5.

Escherichia coli survival

The bacteria grew overnight in LB medium, shaking at 37° C. Then a small amount of this is pipetted into sucrose solutions of varying concentrations. The solutions were then sampled after various timesteps, inoculated into 0.2 mL sterile LB medium and left to grow overnight.

Materials used:

• Pure sucrose
• Tap water
• (Multi-)Pipettes
• E. coli K12 MG1655 strain, upon which the harvard strain and the iJO1366 model are based.
• 2x 96 wells plate (well volume ~200 μL)
• Plate-reader

protocols applied:

• On the day before, inoculate a bottle of LB with E. coli, then let that solution grow overnight at optimal conditions (12 hours). This is our saturated LB solution.
• Under sterile conditions:
• For experiment 1, fill 3 wells from a 96-well plate with 200μL of 0 g/L sucrose solution in sterilized tap water. Do the same for a 100, 200, and 400 g/L sucrose solution.
• For experiment 2, fill 4 wells from a 96-well plate with 200μL of 312.5 g/L sucrose solution in sterilized tap water. Do th same for a 625 g/L sucrose solution.
• For experiment 3, fill 4 wells from a 96-well plate with 200μL of 312.5 g/L sucrose solution in sterilized tap water. Do th same for a 625 g/L sucrose solution.
• Put 200μL LB in each well of an new 96-well plate. Close and keep sterile.
• For experiment 1, innoculate the four different sucrose solutions (three replicates) with bacteria.
• After time steps of 30, 50, 90, and 120 minutes, inoculate the sterile LB-filled wells of with 5 μL of the earlier inoculated sucrose solutions. The final row was inoculated with sterilized sugar water as negative control.
• For experiment 2, innoculate the two different sucrose solutions (four replicates) with bacteria.
• After time steps of 1, 2, 3, 4, 5, and 6 hours, inoculate the sterile LB-filled wells of with 5 μL of the earlier inoculated sucrose solutions. The final row was inoculated with sterilized sugar water as negative control.
• For experiment 3, innoculate the two different sucrose solutions (four replicates) with bacteria.
• After time step of 24 hours, inoculate the sterile LB-filled wells of with 5 μL of the earlier inoculated sucrose solutions. The final row was inoculated with sterilized sugar water as negative control.
• For experiment 1, a plate reader was used to compare overnight growth curves in the LB. For the other experiments bacterial growth was estimated by eye.