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Team:Imperial College London/Protocols Chemotaxis
From 2011.igem.org
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<h1>Phyto-Route</h1> | <h1>Phyto-Route</h1> | ||
<h2>1x PBS</h2> | <h2>1x PBS</h2> | ||
| - | <p>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 1 | + | <p>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 1 litre of PBS:</p> |
| - | <p>- | + | <p>- Disolve following in 800 ml of distilled H<sub>2</sub>O</p> |
<p>- 8 g of NaCl</p> | <p>- 8 g of NaCl</p> | ||
<p>- 0.2 g of KCl</p> | <p>- 0.2 g of KCl</p> | ||
Revision as of 11:42, 19 September 2011
Protocols
This page lists all the protocols used in our project. We have classified them into five main categories as follow.
Phyto-Route
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 1 litre of PBS:
- Disolve following in 800 ml of distilled H2O
- 8 g of NaCl
- 0.2 g of KCl
- 1.44 g of Na2HPO4
- 0.24 g of KH2PO4
- adjust pH to 7.4
- adjust volume 1 L with additional distilled H2O
- autoclave
Note: also possibility to use 1X PBS tablets (one tablet per 200 ml)
Motility medium
Some of chemotaxis assays require cells to be suspended in motility medium. This is recipe for total volume of 100 ml:
- 0.1 g of (NH4)2SO4
- 1.044 g of K2HPO4
- 0.00379 g of EDTA
- if the assay requires bacteria to be suspended in motility medium longer than 40 minutes, 0.4 g of 80% glycerol is added
- autoclave
- after autoclaving add 18 µl of 0.1 M stock solution of FeSO4
Preparation before chemotaxis experiments
This is a procedure required to achieve optimum growth of flagellated bacteria, that will move towards a source and their preparation for chemotaxis assays:
- Add required amount of antibiotic into LB broth (30 ml) before inoculation of bacteria
- Inoculate cells into LB (30 ml) and grow them at 30°C at low shaking 100 rpm overnight
- Centrifuge overnight culture at 5000 rpm for 10 minutes, and resuspend in 2 ml LB
- Inoculate 1 ml of resuspended cells into conical flask with 100 ml LB
- Grow at 30°C and low shaking 150 rpm, until middle of exponential phase is reached
- To obtain cells in mid-exponential phase, 100 µl 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 for the assay
- Take 100ml of mid-exponential phase cell culture and centrifuge it down at 3000rpm for 20 minutes
- Resuspend the centrifuged cells in 10 ml of 1x PBS buffer
- Centrifuge resuspended cells at 3000 rpm for 20 minute
- Resuspend the centrifuged cells in 4 ml of motility medium
Agar plug assay
- 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 20 µl of attractant on to another set of filter paper circles. Position these 2 cm away from the bacterial circle on each of the semi-solid agar plates.
- Leave bacteria to grow in the plates overnight at 30°C
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 a recipe for a total volume of 1 L (dissolved in H2O):
- 12.8 g of (Na2HPO4)7H2O or 6.76 g Na2HPO4
- 3 g of KH2HPO4
- 0.5 g of NaCl
- 1 g of NH4Cl
- Adjust pH to 7.0 - 7.4
- Add 20 ml of 20% glycerol (other protocols might suggest addition of separately sterilised glycerol after autoclaving the salts, I do not do it, it still works)
- 2 g agar
- autoclave
- cool down to 50°C in waterbath and add required antibiotics and separately sterilised solutions
- 2 ml of 1 M filter sterilised MgSO4
- 100 µl of 1 M filter sterilised CaCl2
- Pour plates.
Capillary assay
- Prepare bacteria for chemotaxis.
- Dilute attractant to desired concentrations in motility medium.
- Load a number of capillary tubes (this number depends on the number of attractant concentrations and a number of replicates that is going to be measured) with attractant volumes, if using syringes add 0.1 ml of attractant into each syringe, if using BioRobotix™ tips with ART barrier add 10 µl.
- Set up a structure to hold all the capillary tubes uniformly, add capillary tubes into it and position them, so they relate to the 96 well plate.
- On perfectly leveled surface set up a stand that will hold the rig with all the capillary tubes
- Set up an extendable platform below the rig with capillary tubes
- Add 300 µL of bacterial culture prepared for chemotaxis into separate wells of 96 well plate, filling a number of wells that corresponds to a number of capillary tubes in the rig, fill those wells which relate to capillary tube position.
- Place the 96 well plate with bacterial culture onto an extendable platform, directing wells with bacteria under each capillary tube positioned above, raise the extendable platform so that capillary tubes are inserted in the wells with bacterial suspensions.
- Leave suspended for 40 min at 30°C.
- After 40 min lower the platform and remove 96 well plate with bacterial suspension, position a new 96 well plate underneath the stand with capillary tubes, if using syringes the plate should be empty, if using BioRobotix™ tips with ART barrier, fill a Costar 96 well plate with 290 µl of H2O (the volume can vary, the dilution is necessary for the cell count, I was performing assays with 30:1 dilution).
- Release contents of capillary tubes (attractant + bacteria) into the 96 well plate and in the case of syringe capillaries further move 10 µl into another Costar 96 well plate with dilution of choice (I use 30:1) in H2O.
- Keep on ice and then measure cell count using FACS and obtain data through the Cellquest software.
Behavioral analysis
Plant uptake
- Grow GFP-expressing E. coli to exponential phase
- Spin down bacteria (5000 rpm for 10 min) and remove LB medium supernatant.
- Wash twice with wash buffer (5 mM MES).
- Re-suspend in wash buffer so that the bacteria are at O.D. 30
- Put 10 Arabidopsis seedlings into 100 ml of growth media each
- Add bacteria to plant growth media, add the same amount of wash buffer to the negative control
- Image after 12 h and 24 h
Some notes
- Some departments have very strong restrictions on bringing bacteria into dedicated plant growing areas
- After talking to our health and safety officers, we decided to do all preparation and disposal of bacterial cultures as well as addition of the bacteria to plant media in biosafety hoods in dedicated level 1 or 2 laboratories so that there was no danger of dispersing the bacteria around the plant rooms
