Team:Toulouse/Experiments

iGEM Toulouse 2015

Experiments & Protocols

Transformation Protocol: RbCl method

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 bee hive
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 bee hive
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

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

$$ P\cdot V = n\cdot R\cdot T, \textrm{ideal gaz law} $$ $$ P_A = C_A\cdot R\cdot T = 7,826\cdot10^{-3}\times8,314\times293=19,964 Pa $$

P_A: partial pressure of A in Pa
C_A: Concentration of A in air in mol.m^-3
R: perfect gaz constant = 8,314 J.mol^-1.K^-1
T: temperature in ^°K

$$ P_A = H_A\cdot C_{A,eq}, \textrm{Henry's law} $$ $$ C_{A,eq} = \frac{19,964}{0,019} = 1,019mol.L^{-1}$$

C_A,eq: equivalent concentration in liquid in mol.L^-1
H_A: Henry's constant = 0,019 Pa.m³mol^-1

Transformation Protocol: RbCl method

Media and solution

YETM 500mL	TFB1 200mL	TFB2 200mL
2.5g Yeast Extract 10g Tryptone 5g MgSO4.7H2O Adjust pH to 7.5 with KOH For Plates: add 7.5g of Agar	0.59g KOAc 2.42g RbCl 0.29g CaCl2.2H2O 1.98g MnCl2.4H2O Adjust to pH 5.8 with 0.2 M acetic acid Add dH2O to 200mL Filter sterilize Store refrigerated at 4°C	0.42g MOPS 2.21g CaCl2.2H20 0.24g RbCl 30g Glycerol Adjust to pH 6.5 with KOH Add dH2O to 200mL Filter sterilize Store refrigerated at 4°C

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 5mL of YETM medium. Grow over night at 37°C.
Do not vortex cells at any time after this point in the procedure
3. Dilute 1mL of culture into 50mL YETM medium prewarmed to 37°C

Grow at 37°C for 2hr 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 2000rpm 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 10mL of ice-cold TFB1 with gentle re-pipetting. Use chilled glass or plastic pipette
7. Incubate cells on ice for 5min
8. Repeat step 5
9. Resuspend cells in 2mL 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 200μL volumes into prechilled 0.5mL 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 1,5mL eppendorff on ice: 2-3μL iGEM plate or 1μL plasmid or 10μL ligation.
3. Add 100μL 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
1 µg of	10 µL of miniprep plasmid
1 µL of each restriction enzymes	1 µL of each restriction enzymes
2 µL of Green Buffer	2 µL of Green Buffer
9 µL of Milli-Q water	4 µL of Milli-Q water
Incubate 15 minutes at 37°C

The two parts have a different antibiotic resistance

Both parts
5 µL of miniprep plasmid
1 µL of each restriction enzymes
2 µL of Green Buffer
9 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 0.5x 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
10 µL of insert	no insert
4 µL of vector	4 µL 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
3.5 µL of Milli-Q water	13.5 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 10µL 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