Wednesday, 20 July 2011

Two cultures of wt Arabidopsis thaliana Columbia strain have been set up. Phytogel cultures were set up on Saturday, 15 July and liquid cultures were set up on Wednesday, 20 July.
8 of phytogels are seeded with WT arabidopsis at 1 cm below 1 rim of the plate. Each seed is separated to 1 cm. The phytogel is put under light on the shelves in the planting room to allow the space for roots to grow downwards where we grow the roots for 2 weeks.

Wednesday, 27 July 2011

Preparing 5 x 100 ml MS liquid media, autoclaving the media, put in the cold room and preparing the 2 eppendorf of Venus seedling using the described protocol

Thursday, 28 July 2011

Growing each 20 seeds of Venus in 5 of the prepared 100 ml media by pipetting the seed and transfer the seeds into the media flask under fume cupboard. The flasks are put into the shakers under the light described in the protocol

Friday, 29 July 2011

Start modelling the effect of root branching by auxin. Sending the article involving auxin branching to Nina.
James field preparing GFP bacteria, as supplied by Dr Tom Ellis's lab

Monday, 1 August 2011

Seedling, preparing media and grow more Venus and GFP in 5 x 2 of 100 ml media
Stocking the GFP bacterial culture in glycerol
Start growing bacterial culture in 188 LB media overnight for plant infection tomorrow

Wednesday, 3 August 2011

Today, we prepared new phytogel cultures as the ones we set up previously were getting too poor in nutrients.
We met with Dr Martin Spitaler who advised us on how to prepare samples for the confocal microscopy we will be doing on Friday. The confocal microscopy will focus on imaging GFP expressing bacteria inside Arabidopsis roots to show that uptake of the bacteria takes place. Staining of wt roots with DiD, a lipophilic dye that stains the plant membranes and does not interfere with the absorption or emission spectra of GFP and Dendra, was unsuccessful. However, natural fluorescence was measured in a root in a spectrum that does not interfere with measuring GFP. We should therefore not need to dye the roots before imaging.

Thursday, 4 August 2011

We prepared the GFP-expressing bacteria for plant infection. They were spun down and media was exchanged prior to incubation at 37° to reach exponential phase. Bacteria were then spun down and resuspended in wash buffer (5mM MES) to reach OD 30. 8ml, 4ml and 2ml were added to separate flasks, containing 100ml of half-MS media each. 4ml and 6ml of wash buffer were added to the flasks containing 4ml and 2ml bacteria, respectively. 8ml of wash buffer was added to the negative control. Ten Arabidopsis seedlings were distributed into each of the flasks. Incubation was carried out for 15 hours prior to imaging.

Friday, 5 August 2011

Prior to imaging, roots were washed in PBS to wash off bacteria and facilitate imaging. We imaged the plants incubated with 8ml of bacteria and were able to find bacteria inside one of the roots. A 3D picture was taken of uninfected roots and roots containing bacteria using Z stacking on the confocal micrsocope.

Tuesday, 9 August 2011

Si modelled the concentration of auxin secreted of our bacteria to be 10mM. Accordingly, we used concentrations starting from 10mM to test the effect of different auxin concentrations on the length of the roots and their branching. We made the auxin concentrations by serial dilution and added 10ml of concentrated auxin solution to 100ml of half-MS media each. Twenty-five seeds were added to each flask. The seeds were incubated at 23° and wrapped in aluminium foil to allow the plants to germinate in the dark. They will be allowed to grow in the light in 3 days' time. This follows a protocol described by King et al. (1995).

Team:Imperial College London/Project/Arabidopsis/Notebook

From 2011.igem.org