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From 2011.igem.org

Chemotaxis Lab Protocols

28th of July

Transformation of cells with 6, 7 and 8:
- Let competent cell strain 5α thaw for around 10 minutes on ice.
- Add 2-3μl of DNA.
- Leave on ice for 20-25 minutes.
- Heat shock cells at 42°C for 45 seconds.
- Leave on ice for 10 minutes.
- Add 500μl of LB broth.
- Incubate for 1 hour at 37°C.
- Centrifuge for 1 minute.
- Remove 100μl off the top of the eppendorf tube. Pour out the rest making sure that the pellet remains in the eppendorf tube.
- Re-suspend the cells in the 100μl LB broth solution that was removed in the previous step. - Add 5μl on a chloramphenicol agar plate (concentration of 35μg/ml).
- Add the rest of the sample to a second chloramphenicol agar plate.

Antibiotics:
Four different antibiotics (kanamycin, chloramphenicol, ampicillin & tetracycline) have been used during the course of the project. They have been used in following working concentrations, unless stated otherwise:
- Kanamycin - 35µg/ml
- Chloramphenicol – 35µg/ml
- Tetracycline – 35 µg/ml
- Ampicillin – 100 µg/ml

Tryptone broth
To make bacteria develop flagella they are grown in the tryptone broth. This is recipe for total volume of 1L:
- 10g tryptone
- 1000ml of 1X PBS
- autoclave
- add required amount of antibiotics

1x PBS
Phosphate buffer saline is commonly used in chemotaxis experiments as wash buffer and can also be part of some media. This is a recipe for 1L:
- disolve following in 800ml of distilled H₂O - 8g of NaCl
- 0.2g of KCl
- 1.44g of Na₂HPO₄
- 0.24g of KH₂PO₄
- adjust pH to 7.4
- adjust volume 1L with additional distilled H₂O
- autoclave
Note: also possibility to use 1X PBS tablets (one tablet per 200ml)

Motility medium
Some of chemotaxis assays require cells to be suspended in motility medium. This is recipe for total volume of 100ml:
- 0.1g of (NH₄)₂SO₄
- 1.044g of K₂HPO₄
- 0.00379g of EDTA
- autoclave
- after autoclaving add 18µl of 0.1M stock solution of FeSO₄

5th of August

Preparation before chemotaxis experiments

This is a procedure required to achieve optimum growth of flagellated bacteria that will move towards a source:
- Add required amount of antibiotic into LB broth (30 ml) before inoculation of bacteria.
- Inoculate cells into LB (30ml) and grow them at 30°C at low shaking 100 rpm overnight.
- Centrifuge overnight culture at 5000rpm for 10 minutes, and resuspend in 2 ml LB.
- Inoculate 1ml of resuspended cells into conical flask with 100ml LB.
- Grow at 30°C and low shaking 150 rpm, until middle of exponential phase is reached.
- To obtain cells in mid-exponential phase, 100µl of growing cell culture is taken every 30 minutes and diluted with 900µl LB and absorbance is measured at OD₆₀₀ and graph is plotted. Once the gradient looks exponential (usually around OD₆₀₀ 0.4 - 0.6 after multiplying x10 due to dilution), cells are ready to use.
- Take 100ml of mid-exponential phase cell culture and centrifuge it down at 3000rpm for 20 minutes.
- Resuspend the centrifuged cells in 10ml of 1x PBS buffer.
- Centrifuge resuspended cells at 3000rpm for 20 minutes.
- Resuspend the centrifuged cells in 4ml of motility buffer.

Agar plug in experiment

- Take small circles of filter papers and soak it in the bacterial suspension obtained from the preparation before the experiment and insert into the semi – solid agar plate. Make sure not to insert bacteria too deep into the semi - solid agar since they might start to move using twitching motility on the surface and that is not the desired movement we require during chemotaxis assays.
- Add 20µl of attractant on to another set of filter paper circles. Position these 2cm away from the bacterial circle on each of the semi - solid agar plates.
- Leave bacteria to grow in the plates overnight at 30°C.

18th of August

M9 minimal medium semi - solid agar

In chemotaxis assays semi-solid agar is used as it allows greater diffusion of molecules and allows movement of bacteria within agar. This is recipe for total volume of 1L (dissolved in H₂O):
- 12.8g of (Na₂HPO₄)7H₂O or 6.76g Na₂HPO₄
- 3g of KH₂HPO₄
- 0.5g of NaCl
- 1g of NH₄Cl
- adjust pH to 7.0 - 7.4
- add 20ml of 20% glycerol (other protocols might suggest addition of separately sterilised glycerol after autoclaving the salts, I do not do it, it still works)
- 2g agar
- autoclave
- cool down to 50°C in waterbath and add required antibiotics and separately sterilised solutions
- 2ml of 1M filter sterilised MgSO₄
- 100µl of 1M filter sterilised CaCl₂
- pour plates

Capillary assay

Prepare bacteria for chemotaxis. Load a number of 1ml syringes (this number depends on the number of attractant concentrations and a number of replicates that is going to be measured)

Seedling protocol

- Weigh in appr. 50mg of arabidopsis seeds in eppendorf tube (one tube per 250 ml erlenmayer flask)
- Wash with 500 µl 70% EtOH for appr. 4-5 minutes per tube (mix well)
- Remove 70% EtOH and replace with 500µl 50% bleach
- Incubate for 20 minutes
- Wash several times with sterile ddH2O to remove bleach x3
- Vernalize seeds for 2-3 days

Prepare sterile medium

- Half strength Murashige salt (2.1g per liter ddH2O)
- Add 0.546g MES salt (buffer) per liter of media
- Adjust pH to 5.7-5.8 using 2M KOH
- add 10g sucrose (normally from 1% solution)
- Add 1% agarose = 10g/litre if making phytogel
- Distribute into erlenmayer flasks (125 ml/250ml flask)
- Autoclave for at least 15 minutes

Some notes

- Growth conditions : flasks on a shaker at appr. 200 rpm at constant light conditions
- Grow seedlings for 5-6 days

Auxin uptake protocol

Overview : synthetic auxin is used to see the effect of Arabidopsis's root growth. Variation in auxin concentrations is applied to see the sensitivity of auxin in arabidopsis.

- To test auxin sensitivity, Arabidopsis seeds were sown onto medium as given above and supplemented with 0, 0.00001, 0.0001, 0.01, 1, 100, 10000uM indole-3-acetic acid (IAA).
- Medium preparation and seed sowing occurred under 0.5 pE m-2 sec-l incandescent light to minimize photooxidation of IAA.
- Growing is done at 23°C in darkness for three days
- After 3 days, hypocotyl and root lengths were measured on 10 plantslreplication. Data were normalized to lengths as a percentage of the control treatment and subjected to analysis of variance.
-Plants were transferred to light for a further six days

Some notes

- Concentrations of IAA causing 50% inhibition of root and hypocotyl growth (Isow) ere calculated for each replication by solving regression equations with y = y intercept + 2.

Glycerol stock protocol

- obtain the bacterial pellet from centrifugation
- resuspend the pellet with _microl dH20
- add _microl of 80% glycerol in each eppendorf.
- mix bacteria in 80% glycerol by resuspending the liquid many times

Plant uptake of E coli

-grow GFP+ E coli to exponential phase
-spin down bacteria (5000rpm for 10min) and take off LB media
-wash twice with wash buffer (5mM MES)
-resuspend in wash buffer so that the bacteria are at OD 30
-put 10 Arabidopsis seedlings into 100ml of growth media each
-add bacteria to plant growth media, add the same amount of wash buffer to the negative control
-image after 12h and 24h
Some notes
- Any work involving E coli will take place in the teaching labs and the plant growth room will only be used to grow plants in individual, sealed flasks: E coli will be added to media in the teaching labs and media change will also take place in the teaching labs to ensure containment of the bacteria.
- To ensure that no E coli get into the water ways in the plant rooms, we will dispose of the bacteria in the teaching labs by filling them into flasks, applying vircon and autoclaving the solution

Auxin concentration gradient effect on plants

-prepare half-MS phytogels (see above)
-mark spots 2cm apart from each other where you are going to plant the seeds
-inject auxin dissolved in 70% ethanol at one of these points. The phytogel is very soft so you can inject the solution directly into the gel using a Gilson pipette. Use concentrations of 0.0001, 0.001 and 0.01 mM of IAA.
-seed DR5 reporter line seeds at distances of 2cm, 4cm, 6cm, 8cm from the auxin.

Split-root auxin uptake

-prepare horizontally split plates. Pour regular half-MS phytogel into one half and phytogel containing 0.0001, 0.001 and 0.01mM phytogel into the other half. Pour only regular phytogel into the control plates.
-Take a DR5 reporter line seedling, previously grown in liquid culture and plant with one half of the roots in one half of the plate and the rest of the roots in the other half.

Auxin Lab Protocols

27th of July

The strain of E. coli with the copy of super-folded GFP that was incubated the previous night has shown limited/no growth in the prepared LB broth. This could have been caused by:
-No innoculation
-Kanamycin concentration in LB broth was too high. We had used 85μg/ml.

New protocol:
50μg/ml is the recommended concentration. We will use 35-40μg/ml of kanamycin to speed up growth.

28th of July

Transformation of cells with 6, 7 and 8:
-Let competent cell strain 5α thaw for around 10 minutes on ice.
-Add 2-3μl of DNA.
-Leave on ice for 20-25 minutes.
-Heat shock cells at 42°C for 45 seconds.
-Leave on ice for 10 minutes.
-Add 500μl of LB broth.
-Incubate for 1 hour at 37°C.
-Centrifuge for 1 minute.
-Remove 100μl off the top of the eppendorf tube. Pour out the rest making sure that the pellet remains in the eppendorf tube.
-Re-suspend the cells in the 100μl LB broth solution that was removed in the previous step.
-Add 5μl on a chloramphenicol agar plate (concentration of 35μg/ml).
-Add the rest of the sample to a second chloramphenicol agar plate.

4th of August

In order to make the stock solutions of the Salkowski reagent that was used for the PC method we mixed 100ml of 7.9M sulphuric acid with 1.49g of FeCl2.(4H2O). We called this mixture R1.
In order to make the stock solutions of the Salkowski reagent that was used for the S2/1 method we mixed 100ml of 10.8M sulphuric acid with 0.55g of FeCl2.(4H2O). We called this mixture R2.
The PC method allows us to measure Auxin levels of 0.2μg/ml to 20 μg/ml and is far more specific for IAA rather than other indoles.

Salkowski reagent revised

- 0.811g of anhydrous FeCl₃ is dissolved in 10ml H₂O to obtain 0.5M solution
- add 1ml of FeCl₃ 0.5M solution to 50ml of 35% HClO₄
- store at room temperature in absence of sunlight
- To a 1.0-ml aliquot/supernatant add 2.0 ml of FeCl3-HCl04 reagent
To perform this experiment:
-Mix 500 μl of Trp supplemented LB broth (1 mg/ml) with 500 μl of R1.

-Leave in dark at room temperature for 30 minutes.

-Measure OD at 533nm.

The S2/1 method allows us to measure a range from 2 μg/ml to 200 μg/ml but is less specific for IAA. In order to perform this experiment:
-Mix 500 μl of Trp supplemented LB broth (1 mg/ml) with 1000 μl of R1.
-Leave in dark at room temperature for 30 minutes.
-Measure OD at 554nm.

9th of August

To prepare the E.Coli, media and soil for the survivability experiement:
-Make filter paper discs for soaking the media in.
-Transform E.Coli K-12 5α, with a high-copy plasmid containing a GFP expression.
-Innoculate colonies from the transformants into a 4ml LB media and incubate at 37°C and 225 rpm for a period of 5 hours.
-Place 1g of soil into eppendorf tubes 60 times. Autoclave 30 tubes.
-Pour bacteria into two 2ml eppendorf tubes, centrifuge and empty broth, ensuring pellet remains in the eppendorf tube.
-Re-suspend in 1ml LB.
-Place 10µl each on 60 filter paper discs, and place one in each eppendorf tube with soil in it.
-Add Kanamycin to 400 ml of LB to give concentration of 35μg/ml.
-Add 1 ml of the mixture to 60 separate eppendorf tubes.
-Freeze Media.

Taking Results:
-Remove 6 media eppendorfs from freezer, place in 37°C to melt.
-Remove Filter paper disc from soil tubes and put one each into melted media. Try to shake off the soil from the discs. Red is sterile, blue is non-sterile.
-Incubate at 13:00 at 37°C and 225 rpm for 4 hours.
-Record OD600 of tubes.

22nd of August

To rehydrate synthesised DNA:
- Centrifuge for 4 minutes to ensure DNA is at the bottom of the tube
- Consult the Synthesis Report to work out how much water to add - multiply the yield in nmol by 10, and that value is the volume of water in µl
- Gently pipette up and down, then leave for 5 minutes
- Vortex, pipette up and down, and then leave for a further 5 minutes
- Take 10µl of this DNA solution and add it to 90µl of pure water to make a working solution

Colony PCR
- 20.75 ul H2O
- 2.5 ul Pfu Buffer
- 0.25 ul dNTP
- 0.5 ul forward primer
- 0.5 ul reverse primer
- 0.5 ul Pfu Cx polymerase
Make up master mix of everything but polymerase and set up PCR tubes. Pick colonies from your plate, spot it onto your reference grid plate and then pipette up and down in the PCR tube. The high denaturation temperature of PCR lyses the cells and the DNA is used for template to verify that the colonies contain your plasmid.

Heat Stability Analysis

An experiment to determine the heat stability of fluorescent proteins such as GFP.
Protocol:
- Inoculate 500ml of luria broth with cells and grow overnight.
- Spin culture down and resuspend in 40ml lysis buffer (100mM NaCl, 20mM Tris)
- Lyse in a sonicator and spin down to remove cell debris.
- Take 50ul of crude lysate and heat for 5 hours at the following temperatures (degrees centigrade)
51.9 , 54.5 , 59.1 , 63.7 , 68.1 , 71.9 , 76.5 , 81.1 , 85.1
- Dilute 30ul of heat treated samples in 170ul of lysis buffer and measure fluorescence on plate reader.

To Make a Litre of Super Optimal Broth (SOB)

-20g peptone
-5g yeast extract
-0.6g NaCl
-0.45g KCl
-0.95g MgCl₂
-1.2g MgSO₄

Add the materials to a flask, and then make up to one litre with distilled water.

To make a litre of SOC

Make up a litre of SOB, and then add 3.6g of glucose. The glucose acts as a catabolite repressor.

CPEC assembly

make up 50 ul reactions with 5 ul DNA total of each part to be assembled at equimolar concentrations (ie. make appropriate dilutions)

-33.5 ul H2O

-10 ul Phusion HF buffer

-1 ul dNTP mix

5 ul DNA

0.5 Phusion high fidelity DNA polymerase