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From 2011.igem.org

Chemotaxis 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.

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)

2nd of August

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:
- 5g NaCl
- 10g tryptone
- 2g D-glucose
- 3g agar
- 1000ml H₂O
- autoclave
- before making plates, cool down semi-solid agar to 50^oC in water-bath and add required amount of antibiotics

5th of August

Agar plug in experiment
Preparation before experiment is required to achieve optimum growth of flagellated bacteria that will move towards a source:
- 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.

Experimental procedure:
- Take a small volume of bacteria, which have been grown till mid-exponential phase (5 - 15µl) and insert into the semi – solid agar plate. Preferably insert on one side as attractant will be added to the other. Also it is important not to add bacteria too deep into the agar as bacteria will start to move on the surface of petri dish using twitching motility, however that is not the desired movement we want to observe during chemotaxis.
Add small volume (5µl) of attractant to the other side of the semi – solid agar plates. It is recommended to add different concentrations of attractant to a number of plates for observation of saturation of medium.
Leave bacteria, to grow in the plates for 8 – 12 hours 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 5000g to obtain a pellet.
- Remove the supernatant and resuspend in 3ml 1X PBS buffer for washing.
- Centrifuge again for 20 min. at 5000g, 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.

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.