From 2011.igem.org

Revision as of 18:22, 18 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 L:

- 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 10ml of 1x PBS buffer.

- Centrifuge resuspended cells at 3000rpm for 20 minutes.

- Resuspend the centrifuged cells in 4ml of motility 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 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.

Semi-solid agar

Capillary assay

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 OD 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.