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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.