Team:Toulouse/Experiments
Experiments & Protocols
Protocols for varroa tests
Sampling of varroa
Materials
- Bee hive
- Beekeeper suit
- Gloves
- Smoker
- Dry twigs
- Tweezers
- Big brush
- Small brush
- Petri dishes
Ø x h = 35 x 15 mm
Methods
- Slip beekeeper suit and gloves on and go to beehive
- Fire dry twigs in smoker
- Open bee hive and activate smoker to get bees inside the hive
- Take a frame out the hive and remove bees with big brush and smoker
- Close beehive
- In the lab, put the frame on a table against the wall
- With tweezer drill hole into one beehive cell
- Remove larvae and look for varroas on larvae and on beehive cell
- If there are varroas, take them with a small brush and put them on Petri dishes
- Make sure there are two or three larvae on Petri dishes in order to allow survival of varroas
- Start again step 7 to 9 until you have enough varroas
Standardization of varroas and sampling
When we take varroas directly from frame, as it is described in protocol “Sampling Varroas”, we have varroas in different phases. In order to have varroas in the same phase it is necessary to add one step and it is important for reproducibility of experiments. With this method we place varroas on adult bees so all varroas will be in phoretic phase.
Materials
- Bees in box with aeration and glucose
- Varroas from protocol “Sampling varroas”
- Gas cylinder of CO2
- Small brush
- Tweezers
- Petri dishes
Ø x h = 35 x 15 mm
Methods
- With small brush take varroas from Petri dish and put them on bees in box through aeration holes
- Place the box in a 35 °C incubator overnight. Make sure you have a bowl with water in order to have enough humidity in incubator
- Take the box out of incubator
- Add CO2 from gas cylinder into the box until all bees fall down
- Open the box, take a bee with tweezer and look for varroas
- When you find a varroa take him with small brush and replace bee in the box
- Start again step 5 and 6 until you have enough varroas
Attraction test on varroas
In order to test the attraction effect of butyric acid on varroas an Y test was used, as it is showed with diagram below. Where we place varroas for the test we choose glass pipe because on plastic they could load themselves with electrostatics and die. For butyric acid concentration we choose 4% (V/V) because this is the concentration used in the patent we see, see “Attribution” part.
Materials
- Pump wich expels air
- 15 mL Flacon tube
- Plastic pipe
Ø = 10 mm - Glass T pipe
Ø = 10 mm, made by a glassworker - Plastic separator
- Carded cotton
- Absorbent cotton
- 5mL 4% (V/V) Butyric acid
- 5mL Water
- Standardized varroas
Methods
- Put a cotton on Petri dish and add 400µL of one acid formic solution
- Place three varroas on this Petri dish and close it
- Start again step1 and 2 for each formic acid solution and water
- Each 30 minutes check if varroas are alive. To do that :
- When varroa heads for one side of Glass T tube and covers more than 2 cm test is over and we write down the side choosen by varroa (Butyric acid or Water)
- Two tests can be made in the same time thanks to the plastic separator
Mortality test on varroas
To test toxicity of formic acid on varroas we had to choose which concentrations we use. For that we based our thoughts on present treatments. When beekeepers use formic acid for long treatment they place a diffuser at the top of the hive and formic acid concentration was assessed at 200ppm 1 on average that is equivalent to 7.826 mmol.m-3. As gas concentration is difficult to evaluate we calculate the liquid concentration balance thanks to the ideal gas law and the Henry’s law. To simplify calculation we noted down formic acid A.
$$ P\cdot V = n\cdot R\cdot T, \textrm{ideal gaz law} $$ $$ P_A = C_A\cdot R\cdot T = 7,826\cdot10^{-3}\times8.314\times293=19,964 Pa $$
- PA: partial pressure of A in Pa
- CA: Concentration of A in air in mol.m-3
- R: perfect gaz constant = 8.314 J.mol-1.K-1
- T: temperature in °K
$$ P_A = H_A\cdot C_{A,eq}, \textrm{Henry's law} $$ $$ C_{A,eq} = \frac{19,964}{0,019} = 1,019mol.L^{-1}$$
- CA,eq: equivalent concentration in liquid in mol.L-1
- HA: Henry's constant = 0,019 Pa.m3mol-1
Materials
- Petri dishes
Ø x h = 35 x 15 mm - Varroas form “Sampling varroas”
- Cotton
- Acid formic solutions :
- 2 mol.L-1
- 10 mmol.L-1
- 1 mmol.L-1
- 500 µmol.L-1
- 50 µmol.L-1
- Water
Methods
- Put a cotton on Petri dish and add 400µL of one acid formic solution
- Place three varroas on this Petri dish and close it
- Start again step1 and 2 for each formic acid solution and water
- Each 30 minutes check if varroas are alive. To do that :
- Tap on Petri dish and see if varroa move. If yes varroa is alive, if not see below
- Look at binocular magnifier if varroas move their feets. If yes varroa is alive, if not varroa is dead
Remark: A varroa stop moving approximately one or two hours before it dies
Protocols for culture tests
Cytotoxicity tests
Choice of concentrations
In the begining we tested high and low concentrations and in function of results we adapted concentrations. In the end we worked with these concentrations:
- Butyric acid : 218 mM, 109 mM, 10.9 mM, 5.45 mM and 1.09 mM
- Formic acid : 100 mM, 10 mM, 1 mM 500 µM, 100 µM, 50 µM and 25 µM
Materials
- Optical reader, OPTIMA MARS Analysis
- 48 wells plates
- Pre-culture of E. coli BW 25113
- Acid solutions
- Medium : LB, M9 15 mM of glucose or 30 mM of glucose
Methods
- Add 400 µL of medium in each well
- Add 50 µL of pre-culture
- Add 50 µL of acid solution
- Place the 48 well plate in the optical reader
- Adjust parameters on computer.
Usually we set 250 cycles around 24 hours so we have an OD measure every six minutes
Remark: Each condition is tested in three replicates
Growth culture
Culture on erlenmeyers and TubeSpin® Bioreactors
Inoculation and sampling
Materials
- Pre-culture of E.Coli BW 25113 in LB
- Spectrophotometer
- 1mL Spectrophotometer cuvettes
- Centrifuge
- Erlenmeyers
- TubeSpin® Bioreactors from TPP brand
- Medium : M9 15 mM of glucose or 30 mM of glucose
- Incubators at 37 °C, 130 rpm and without agitation
- 1.5 mL Eppendorf
- 0.2 µm filters
Methods
- Add 50 mL of medium on erlenmyer and TubeSpin® Bioreactor
- Inoculate from pre-culture to have OD600nm=0.1.
To do that centrifuge the appropriate volume of pre-culture, then remove LB medium and resuspend sediment with M9 medium to inoculate.
Remark: This step permits to eliminate substrates from LB medium which could interfere during NMR analysis. - Place erlenmeyers in incubator 37°C 130rpm and TubeSpin® Bioreactor in incubator 37°C without agitation
- Sampling every two hours the first day:
- Take 1mL of culture in 1.5 mL Eppendorf.
Attention: for TubeSpin® Bioreactor use needle and syringe in order not to let air enter, as possible. - Add 100 µL of sample in spectrophotometer cuvette, complete with 900µL water and measure OD600nm with spectrophotometer
- Centrifuge the rest of samples at 13000 rpm during 3 minutes
- Filter the supernatant through a 0.2 µm filter and conserve it at -20°C
- Days follow sample once a day with method below and plate on Petri dish an appropriate dilution in order to know if bacteria are alive
NMR analysis
Materials
- Culture supernatants from -20°C
- 2.5 mM TSP (Trimethylsilyl propanoic acid) diluted in D2O
- 0.5 mm NMR tubes
- 1.5mL Eppendorf
- Spinners (5mm)
- 500MHz Bruker Avance Spectrometer
Methods
- Add 400 µL of culture supernatant in 1.5mL Eppendorf
- Add 100 µL of TSP solution
- Place the mix in 0.5 mm NMR tubes
- Place NMR tube into spinner, sample is ready to analyse
Culture on 48 wells plates
In order to determine the right concentration of polysaccharide and enzyme of BioSilta kit we have to do several cultures at the same time. So, we use an optical reader and 48 wells plates in order to have lots of test in the same time.
Materials
- Optical reader, OPTIMA MARS Analysis
- 48 wells plates
- Pre-culture of E. coli BW 25113
- Different concentrations of BioSilta medium
- For one concentration of BioSilta medium different concentrations of enzyme
Methods
- Add 450µL of medium in each well
- Add 50µL of pre-culture
- Place the 48 well plate in the optical reader
- Adjust parameters on computer. We tested culture between one day and ten days
Remark: Each condition is tested almost in two replicates. According to our results we adapt concentrations of Biosilta medium and enzyme, results are exposed in Device part.
Enzyme kinetic
Materials
- Spectrophotometer
- BioSilta medium
- BioSilta enzyme solution named Reagent A (3000U/L)
- Bradford’s reagent
- 1.5mL Eppendorf
- Standard solutions of glucose
Methods
- For each standard solutions : in a 1.5 mL Eppendorf sample 10 µL and add 1mL Bradford’s Reagent, wait 20 minutes and measure OD505nm
- Plot glucose concentration in function of OD505nm and determine the linear region
- Add 1.5 mL of BioSilta medium and 45 µL of reagent A (50 U/L) in an Eppendorf
- Sampling every 30 minutes:
- Take 10 µL and add 1 mL of Bradford’s reagent in an Eppendorf
- Wait 20 minutes
- Measure OD505nm
- If OD505nm is over linear region dilute sample and measure OD505nm again
- Stop sampling when glucose concentration no longer change
Acids production test
In order to test if E.coli produce formic acid and butyric acid with genes we add we made culture test with modified bacteria. We used the same protocol as “Culture on Erlenmeyers and TubeSpin® Bioreactors” with some changes:
- Volume of culture : 30mL
- Add Ampicillin at 25µg/mL to have selection pressure
- The number of samples:
- Sample at the beginning
- Sample at the end of the first day
- Sample after 24h culture and 48h culture
Test of gas concentration
The objective of our device is to produce gas, so we would like to know gas composition of our culture. So, we developed a system in order to recover acids gas.
Materials
- 4 h culture in 50 mL Falcon in M9 medium with 15 mM of glucose
- Silicon plugs adapted to 50mL Falcon
- Needles
- 0.2 µm filters
- 10 mL Syringes
- Neoprene pipes Ø=0.8 mm
- 10 mM NaHCO3
- 1.5mL Eppendorf
- 1mL Sterile cone
- Incubator 3°C without agitation
Methods
- Replace Falcon plug with silicon plug
- Adjust fliter on needle and peg it into silicon plug. Do it twice
- Adjust neoprene pipe into each filter
- Add 700µL of NaHCO3 in an Eppendorf
- At the end of first pipe put a sterile cone and immerse it into Eppendorf with NaHCO3
- At the end of second pipe put a 10mL syringe
- After 24h culture, press 10mL syringe in order to expel gas in NaHCO3 solution
- Conserve samples at -20°C before NMR analysis (see protocol foregoing)
Remark 1: We used culture in M9 because with the “Acids production tests” we had data on this medium.
Remark 2: 10 mM NaHCO3 solution was used because pH was 8.3 so it would permit acid gas solubilisation.
Protocols for TPX permeability tests
Preparation of TPX bag
Materials
- TPX, gas permeable plastic
- Fusing machine
- 2 mM Formic acid solution
- 4 % (V/V) Butyric acid solution
Methods
- Prepare plastic bag in sticking on 3 sides over 4 with fusing machine
- Add 7mL of appropriate solution in plastic bag
- Stick on the last side with fusing machine
Permeability test
To test gas permeability of TPX plastic, we use the same protocol as “Test of gas concentration”. The only change is that no filters were used because the sterility is not necessary.
The device used for the permeability test
Transformation Protocol: RbCl method
Media and solution
| YETM 500 mL | TFB1 200 mL | TFB2 200 mL |
|---|---|---|
|
|
|
Preparation of Competent Cells
- 1. Streak cells froms frozen stock onto YETM plate. Incubate overnight at 37 °C
- 2. Pick a single fresh colony to inoculate 5 mL of YETM medium. Grow over night at 37 °C.
- Do not vortex cells at any time after this point in the procedure
- 3. Dilute 1 mL of culture into 50 mL YETM medium prewarmed to 37 °C
- Grow at 37 °C for 2 hours with agitation
- Volumes can be scaled up 5X and all of the 5mL overnight culture can be used
- 4. Transfer culture to sterile 50 mL tube. Chill on ice/water 10-15 minutes
- 5. Centrifuge for 10 minutes at 2,000 rpm at 4 °C. Immediately aspirate off all of the supernatant
- Do not allow cells to warm above 4 °C at any time in this procedure
- 6. Resuspend cells in 10 mL of ice-cold TFB1 with gentle re-pipetting. Use chilled glass or plastic pipette
- 7. Incubate cells on ice for 5 minutes
- 8. Repeat step 5
- 9. Resuspend cells in 2 mL of ice-cold TFB2 with gentle re-pipetting. Use micropipet tip (plastic)
- 10. Incubate cells on ice for 15 minutes
- Cells may be used for transformation or frozen
- To freeze: aliquot cell in 200 μL volumes into prechilled 0.5 mL microfuge tube (on ice)
- Freeze immediately in liquid nitrogen
- Store cells frozen at -80 °C
Transformation of Competent Cells
- 1. If starting with frozen competent cells, warm tube/cells by gently twirling between your fingers until just thawed.
Immedately place on ice for about 5 minutes - 2. Add to 1,5 mL eppendorff on ice: 2-3 μL iGEM plate or 1 μL plasmid or 10 μL ligation.
- 3. Add 100 μL of competent cells and mix by gentle re-pipetting
- 4. Incubate cells on ice for 20-30 minutes
- 5. Heat shock the cells exactly 90 seconds at 42 °C
- 6. Return cells on ice for 2 minutes
- 7. Add 1mL of YETM medium. Incubate at 37 °C for 45-60 minutes with slow gentle shaking
- 8. Plate 0.1-0.2 mL of transformed cells on LB-plate containing the appropriate antibiotic. Incubate overnight at 37°C
Minipreps
- 1. Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C, …)
- 2. Resuspend one colony per culture tube in 5 mL of LB medium with antibiotic
- 3. Let the culture grow overnight at 37 °C in a shaking incubator
- 4. Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with elution buffer or water at 55 °C
- 5. Keep the tubes at -20 °C
Cloning
First step: Digestion
Both parts have the same antibiotic resistance
| Vector | Insert | Digestion control first enzyme | Digestion control second enzyme |
|---|---|---|---|
Volume equivalent to 1 µg of vector miniprep |
Volume equivalent to 1 µg of insert miniprep |
Volume equivalent to 1 µg of vector miniprep |
Volume equivalent to 1 µg of vector miniprep |
1 µL of each restriction enzymes |
1 µL of each restriction enzymes |
1 µL of the first restriction enzyme |
1 µL of the second restriction enzyme |
2 µL of Fast Digest Green Buffer (Thermo Scientific™) |
2 µL of Fast Digest Green Buffer (Thermo Scientific™) |
||
Up to 20 µL of Milli-Q water |
Up to 20 µL of Milli-Q water |
||
Incubate 15 minutes at 37 °C |
|||
The two parts have a different antibiotic resistance
| Both parts |
|---|
Volume equivalent to 1 µg of DNA miniprep |
1 µL of each restriction enzymes |
2 µL of Fast Digest Green Buffer (Thermo Scientific™) |
Up to 20 µL of Milli-Q water |
Incubate 15 minutes at 37°C |
Migration and gel extraction
- 1. Prepare a 1 % or 2 % electrophoresis agarose gel with 0.5 X TAE buffer
- 2. Put 20 µL of sample + 6 µL of marker (1 kb for 1 % gel and 100 pb for 2 %) into the well
- 3. Migration for 30 min at 100 V or 1 hour at 50 V
- 4. Bathe 10 minutes in BET
- 5. Wash in water for 5 minutes
- 6. The gel extraction is realized thanks to the QIAGEN Gel Extraction Kit
- Two ways to inactivate the enzymes for the vector
- Use of DNA Clean up kit for the DNA fragment above 200 pb
- Heat inactivation at 95 °C for 10 minutes
Second step: Ligation
| Mix | Control |
|---|---|
Volume equivalent to 3 molecules of insert (for one molecule of vector) |
no insert |
Volume equivalent to 50 ng of vector |
Volume equivalent to 50 ng of vector |
2 µL of 10X T4 buffer |
2 µL of 10X T4 buffer |
0.5 µL of T4 ligase |
0.5 µL of T4 ligase |
Up to 20 µL of Milli-Q water |
Up to 20 µL of Milli-Q water |
Incubate the ligation mix 15 minutes at room temperature (22°C) |
|
Keep the tubes in ice or at -20 °C to prepare the transformation |
|
Third step: Ligation
- 1. Take 10 µL of the ligation mix for 100 µL of competent cells and use the Toulouse iGEM Team 2015 transformation protocol
- 2. Plate the solution on selective medium overnight at 37 °C
References
