From 2011.igem.org

Latest revision as of 18:34, 27 October 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:

- Dissolve the 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 to 1 L with additional distilled H₂O.

- Autoclave.

Note: it is also possible to use 1X PBS tablets (one tablet per 200 ml).

Motility medium

Some chemotaxis assays, that are based on metabolism generated attractant gradient, require cells to be grown in motility medium. This is a recipe for total volume of 1 L:

- 20 ml of 20% glycerol (other protocols might suggest addition of separately sterilised glycerol after autoclaving the salts, I do not do do that and it still works)

- 2.0 g of (NH₄)₂SO₄

- 11.2 g of K₂HPO₄

- 4.8 g of KH₂PO₄

- Autoclave

- After autoclaving add 0.25 g of MgSO₄ and 0.25 mg of FeSO₄.

Wash buffer

This buffer is used during the preparation of bacteria for chemtoaxis and can also be used as chemotaxis buffer during capillary assays. This is recipe for 100 ml:

- 0.136 g of KH₂PO₄

- 0.174 g of K₂HPO₄

- 3 mg of EDTA

- Control pH and adjust to be in between 7.0 and 7.4 pH.

Some notes

- Addition of EDTA is absolutely necessary as it removes any heavy metal ions, which can inhibit chemotactic behaviour and motility altogether.

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 a 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 900 µl LB and absorbance is measured at O.D.₆₀₀ and a graph is plotted. Once the gradient looks exponential (usually around O.D.₆₀₀ 0.4 - 0.6 after multiplying x10 due to dilution), cells are ready for the assay.

- Take 100 ml of mid-exponential phase cell culture and centrifuge it down at 3000 rpm for 20 min.

- Resuspend the centrifuged cells in 10 ml of Wash buffer.

- Centrifuge resuspended cells at 3000 rpm for 20 minutes.

- Resuspend the centrifuged cells in 4 ml of Wash buffer.

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 do that and it still works).

- 2 g agar

- Autoclave.

- Cool down to 50°C in a water bath and add required antibiotics and the following 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 wash buffer.

- Load a number of 200 µl pipette tips (this number depends on the number of attractant concentrations and a number of replicates that is going to be measured) with 10 µl of attractant into each tip. Remove pipette from the tip, while keeping the tip still submerged in the eppendorf tube with the attractant. Then seal the top of the pipette tip, making sure that the volume inside the tip does not change. Once sealed, the tip can be taken out of the eppendorf without losing the attractant from the tip.

- Set up a structure to hold all the tips uniformly, add the tips into it and position them, so they relate to the 96 well plate.

- On a perfectly level surface set up a stand that will hold the rig with all the tips containing different attractant concentrations.

- Set up an extendable platform below the rig with the tips.

- 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 tip positioned above, raise the extendable platform so that the capillary tubes are inserted in the wells with bacterial suspensions.

- Leave suspended for 60 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, with 290 µl of wash buffer in each of the wells, which will collect the attractant with bacteria from the tips (the volume can vary, the dilution is necessary for the cell count, I wanted this to be 30:1 dilution).

- Release contents of capillary tubes (attractant + bacteria) into the 96 well plate and then move to another plate, which contains 150 µl of wash buffer, 150 µl of bacterial suspension diluted 30:1 to obtain final dilution of 60:1.

- Use the 96 well plate with 60:1 dilution to measure cell count using FACS and obtain data through the Cellquest software.

- Also from the 96 well plate containing 30:1 dilution with bacteria obtained from the tips, a number of different dilutions can be performed so that CFU (colony forming unit) count can be performed to obtain data. (I use two final dilutions 300:1 & 15000:1).

Some notes

- When collecting data using BD FACScan, it is advisable that the bacteria flouresce. It eases gating of the appropriate cells and subsequent data collection.

Live imaging of bacterial movement

- Grow overnight culture of bacteria in 5 ml LB in 15 ml falcons at 30°C and 200 rpm.

- Prepare attractant or control. For control we have used motility buffer and for attractants we have used 10 mM serine in the motility buffer and 10 mM malate in motility buffer.

- Use 180 µL of overnight culture to inoculate into 10 ml of LB in 50 ml falcons.

- Grow bacteria until early exponential phase (OD₆₀₀ 0.35).

- Once in required OD₆₀₀ separate the volume into same 1.5 ml aliquots and spin cells down for 10 min at 2000 rpm.

- Resuspend the bacterial pellets in 0.5 ml of motility buffer with glycerol and leave at 30°C and 150 rpm for 2 hours.

- Prepare samples by diluting cells 1:20 to studied attractant or control and add into 35 mm microscopy petri dishes with coverslip bottom.

- Observe for 5 min at 30°C using Zeiss Axiovert 200 Inverted Fluorescent Microscope and collect video using Volocity software with frame caption 1 frame per second.

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.