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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 1L:

- disolve following 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)

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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 100ml:

- 0.1g of (NH4)2SO4

- 1.044g of K2HPO4

<- 0.00379g of EDTA - autoclave - after autoclaving add 18µl of 0.1M 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.