From 2011.igem.org

Latest revision as of 12:05, 28 October 2011

Protocols

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

Plant

Seeding

Vernalise seeds (this has to be done 2 to 3 days before seeding!)
- Weigh approx. 50 mg of Arabidopsis seeds in eppendorf tube (one tube per 250 ml Erlenmayer flask).
- Wash with 500 µl 70% EtOH for approx. 4-5 minutes per tube (mix well).
- Remove 70% EtOH and replace with 500 µl of 50% bleach.
- Incubate for 20 min.
- Wash 3x with sterile ddH2O to remove bleach.
- Vernalize seeds for 2-3 days.

Some notes
- Growth conditions : flasks on a shaker at approx. 200 rpm in constant light conditions.
- Grow seedlings for 5-6 days.
- For data fitting experiment, 2 set of phytogels are used, to be able to measure the IAA degradation rate and the length of the root at the same time.

Uptake

Overview : synthetic auxin is used to see the effect on Arabidopsis root growth. Variation in auxin concentrations is applied to see the sensitivity of auxin in Arabidopsis.

- To test auxin-sensitivity, Arabidopsis seeds were sown onto medium as given above and supplemented with 0, 0.00001, 0.0001, 0.01, 1, 100, 10000 µM indole-3-acetic acid (IAA).
- Medium preparation and seed sowing occurred under 0.5 pE m-2 sec-l incandescent light to minimize photo-oxidation of IAA.
- Growing is done at 23°C in darkness for 3 days.
- After 3 days, hypocotyl and root lengths were measured on 10 replicate plants. Data were normalized to length as a percentage of the control treatment and subjected to analysis of variance.
- Plants were transferred to light for a further 6 days.

Some notes
- Concentrations of IAA causing 50% inhibition of root and hypocotyl growth (Isow) were calculated for each replication by solving regression equations with y = y intercept + 2.

Making phytogels/ liquid media

Half-strength Murashige salt (2.1 g/L ddH2O)
- Add 0.546 g MES salt (buffer) per liter of media.
- Adjust pH to 5.7-5.8 using 2M KOH.
- Add 10 g sucrose (normally from 1% solution).
- Add 1% agarose = 10 g/L if making phytogel.
- Distribute into Erlenmayer flasks (125 ml/250 ml flask).
- Autoclave for at least 15 min.

Seeding Arabidopsis into soil in soil erosion experiment

- Set up the experiment with 18 (3 columns at 10 cm side x 6 rows on 20 cm side) where Arabidopsis are seeded in 3 rectangular pots which represented each concentration of IAA. Together with 2 controls (one where no IAA was watered and one where no Arabidopsis was seeded), 30 pots in total.
- Treat 12.5 kg of the M2 soil with 1 L of 1 mM fungicide.
- Rest the soil overnight.
- Fill the soils in the container and water each 1 kg of soil in 100 ml of water.
- Make a 1 cm depth hole into the soil.
- Pipette each seed to the center of each hole.
- Cover the container with a plastic film to prevent water evaporation.
- Water with the same amount of IAA concentrations everyday for 3 days. After that water once every 2 days.

Obtaining the water mass retained in soil erosion experiment

- At 10 minutes after final watering, 10 cm³ of soil at a region between 4 plant roots is collected for measuring wet mass.
- The soil is incubated for 1 day to get rid of water to achieve dry mass. The difference between the wet and the dry mass was the water hold up in the soil.
- The plants are left 2 days with no watering and the same procedure to measure water hold up was repeated. The smaller the difference of water retained between 10 min and 2 days after watering, the more water was retained by the soil