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From 2011.igem.org

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Protocols

This page lists all the protocols used in our project. We have classified them into five main categories as follow.

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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)

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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

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Preparation

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Agar plugin

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Semi-solid agar

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Capillary assay

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Plant uptake

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- 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.