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Team:Imperial College London/Project/Arabidopsis/Notebook
From 2011.igem.org
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 protocolThursday, 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 protocolFriday, 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 mediaStocking 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.The response of Arabidopsis to different concentration of synthetic auxin (Monday 8 and Tuesday 9 August 2011)
Auxin/IAA is a main hormone important to root growth and root branching. Different level of IAA concentration acts differently on Arabidopsis. From Joseph et al 1995, increasing exogenous IAA concentration from 0 - 0.1 nM increases the root growth from 0 - 20%. From 0.1 nM to 10microM, the root length increases sequentially but the fibrousity of root increases until the plant die at more than 10 microM. However ... tells that the optimal auxin concentration is in between 0.5 microM - 20 microM. The modelling team on the other hand suggest the model they propose suggest the optimal concentration of 10 mM. Therefore the concentrations used in these experiments vary from 10 mM, 0.1 mM, 1 microM, 10 nM and 0.1 nMThe response of Arabidopsis to different distances from where synthetic auxin is applied in phytogel (Wednesday 10 August) 2011
Even though E.coli is engineered to be uptaken into the root to switch on the production of IAA inside Arabidopsis, however another theory is proposed whether E.coli could be engineered to produce IAA outside the root and let the IAA diffuses into the root directly. The arabidopsis strain we use is Venus which could produce yellow fluorescence protein under the presence of the IAA binding to the YFP promotor. Within the limit period of time the length of root is measured and the endogenous IAA level is estimated by the intensity of fluorescence resulting from YFP under the confocal microscope. The experiment is conducted in phytogel in order to simulate the release of auxin from E.coli into a soil near the plantThe concentrations we use here follow ... which are 0 microM (control), 0.1 microM, 1 microM and 10 microM. Preparation of each concentration is carried on through 10x serial dilution from 1 mM IAA in 70% ethanol from 10 mM IAA solution stock. We then divide 20 phytogel plates, 5 of each with the same concentration in order to increase the number of data. At each plate, the seed is sown at distances of 2,4,6,8, and 10 cm away in x-direction from the point where 0.1 ml of IAA concentration is applied. The space in y-direction is allowed for the root to grow from each seed. The plate is kept in low amount of light for 3 days in order to prevent photooxidation of auxin and also in 4 C to simulate the winter hibernation. The plates are later put in light for another 6 days to see the root growth. The protocol for preparing phytogel, seedling, and growing plant phytogel is found in the protocol section.
