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Wageningen UR iGEM 2016

Overview

Description

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

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In Vitro Assay

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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!

Cas9-based 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:

Ingredient	Amount
5x Q5 buffer	5 µL
dNTP mix (10 mM)	0.5 µL
Q5 polymerase	0.25 µL
fwd primer	100 ng
rev primer	100 ng
template	10-100 ng
dH₂O	to 25 µL

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	Temperature in °C	Time
1	98	30 seconds
2	98	30 seconds
3	55	1 minute
4	72	9 minutes
5	4	∞

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(hyperlink) 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-pAzF(ref) containing the arabinose promoter and CAT resistance gene.

Primers to generate fragments for yeast assembly were made using the href=”http://nebuilder.neb.com/”>NEBuilder for Gibson assembly, with 40 bp overlap between fragments. PCR was performed as usual (hyperlink), 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

Ingredient	Concentration
YNB (Sigma Aldrich)	6.7 g/L
UDM (Yeast Synthetic Drop-out Medium Supplements w/o Uracil; Sigma Aldrich)	1.92 g/L
Glucose	20 g/L
Agar	20 g/L (for plates)

10x TE

Ingredient	Concentration
Tris	10 mM
EDTA	1 mM

pH was adjusted to 7.5.

10x LiAc

Ingredient	Concentration
Lithium Acetate	1 M

pH was adjusted to 7.5 and filter sterilized.

STET buffer

Ingredient	Concentration
sucrose	8%
Tris pH 8	50 mM
EDTA	50 mM
Triton X-100	5%

Cloning of gRNA plasmids using annealed oligos

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

Oligos were annealed as in Jao et al(ref), 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 (hyperlink), 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 (hyperlink).

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:

Ingredient	Amount
10x reaction buffer	1.5 µL
NTP	1.5 µL each
Digested template DNA	1 µg
T7 RNA polymerase mix	1.5 µL
dH₂O	to 20 µL

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 whiteish 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

Ingredient	amount
Tris base	54 g
Boric acid	27.5 mL
EDTA (0.5 M, pH8)	20 mL
MQ	to 1 L

pH should be ~8.3.

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

Ingredient	Amount
Urea	10.5 g
MQ	6.25 mL
40% acrylamide	6.25 mL
5x TBE	5 mL

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

Ingredient	Amount (mL)
Formamide	9.5
EDTA (200mM)	0.25
10% SDS	0.025
2.5% BPB	0.1
2.5% CFF	0.1

RNA elution buffer

Ingredient	Concentration
NaCl	0.5 M
MgCl₂	10 mM
EDTA	1 mM
SDS	0.1%

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 (hyperlink) 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

Ingredient	Concentration
KPi	50 mM
NaCl	300 mM

pH was adjusted to 8.0.

His buffer B

Ingredient	Concentration
KPi	50 mM
NaCl	300 mM
Imidazole	500 mM

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:

Ingredient	Amount
5x Cas9 assay buffer	6 µL
Streptococcus pyogenes Cas9 (1µM)	1 µL
gRNA	~90 ng/guide
dH₂O , nuclease free	to 28 µL

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 instead (Thermo Fisher Scientific) for ~25 minutes instead of SybrSafe.

5x Cas9 assay buffer

Ingredient	Concentration
HEPES	100 mM
NaCl	500 mM
MgCl₂	25 mM
EDTA	0.1 mM

pH was adjusted to 6.5.

References

1.

Bacillus thuringiensis

↩

2.

In vitro

varroa destructor

Apis mellifera