In collaboration of IGEM oxford Team we managed to do a toxicity assay of Curldan as we want to use it in vineyards. Our lab does not have the required material to do so and this would help complete our project. So we sent them a sample of our produced Curdlan so they could do tests on it. They decided to test curdlan toxicity on two Bacteria, Rhodobacter sphaeroides and Pseudomonas putida. They choose these bacteria because these two species are very common in the environment so they believe these bacteria represent a good marker for an eventual environmental toxicity.
Methods and Materials
Single colonies of wild-type Rhodobacter sphaeroides WS8N and Pseudomonas putida KT2440 were grown in SUX media and Lysogeny Broth respectively overnight to stationary phase at 30°C with orbital shaking at 225 rpm. 1% w/v curdlan in NaOH was made by adding 0.1g of solid curdlan to 10mL of 0.5M NaOH and warming the mixture at 55°C with regular vigorous shaking for 15 minutes to achieve complete dissolution. 100µL of stationary culture was added to 100µL of curdlan-NaOH or NaOH in the wells of a Corning 96-well flat bottom plate, and each condition was repeated in six-plicate. The plate was incubated at 30°C in a FLUOstar Omega Microplate Reader with 200 rpm shaking in between reads, and OD600 was monitored every 15 minutes. The data obtained from each six-plicate was averaged, and error bars were constructed using the standard deviations at each time point.
Conclusions
- 1% curdlan does not appear to present any additional toxicity over the 0.5M NaOH solvent. - Curdlan seems to be not toxic for our markers.
Perspectives
This assay is a good news for us but we have to go deeper into our safety tests. A good way to do that is to test the toxicity of our curdlan on Mammal cells and test a bigger quantity of curdlan (up to 10%)
Throughout the year, we have been contacted to answer surveys and to share surveys. We answered to the best of our abilities all of the following surveys: UI-Indonesian , UCL , TEC-Monterry. When possible we gave feedback to improve their surveys. We also participated to Paris-Bettencourt's rhizi project , which is a good method to connect different iGEM teams and collaborate