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 instead of LB broth. This is recipe for total volume of 1L:
- 10g tryptone
- 1000ml of 1X PBS
- autoclave
- add required amount of antibiotics
To make 1X PBS (phosphate buffer saline):
-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.
12th of August
Swarm plate assay
Preparation of semi solid agar with attractant:
- Make semi solid agar normally
- During pouring of the plates, in addition antibiotic add appropriate amount of attractant to the plate (5mM)
Preparation of bacteria before the experiment:
- Add required amount of antibiotic into tryptone broth (TB 5 ml) before inoculation of bacteria.
- Inoculate cells into TB (5ml) and grow them at 30°C at low shaking 100 rpm overnight.
- Take 150µl of overnight cell culture into fresh 5ml TB (with correct amount of antibiotic).
- Grow at 30°C and low shaking 100 rpm, until middle of exponential phase (expected OD600 around 0.35 - 0.4), this can take up to 4-6 hours.
- Centrifuge cells for 20 minutes at 5000rpm to obtain a pellet.
- Remove the supernatant and resuspend in 3ml 1X PBS buffer for washing.
- Centrifuge again for 20 min. at 5000rpm, once done remove supernatant and resuspend cells in 5/6ml TB, with desired OD600 around 2.5.
Experimental procedure:
- Take 200 - 500µl of resuspended cells, and insert it into the semi solid agar.
- Incubate at 30°C for ... hours and observe concentric rings.
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 1 litre of 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 required of separately sterilised solutions
- 2ml of 1M filter sterilised MgSO4
- 100µl of 1M filter sterilised CaCl2
- pour plates
Capillary assay
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.
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