Oct 26, 2020
DNT Detection In Soil
- 12020 iGEM NEFU China
- 2020 iGEM NEFU China
Protocol Citation: Zhujun Wei 2020. DNT Detection In Soil. protocols.io https://dx.doi.org/10.17504/protocols.io.bnydmfs6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 26, 2020
Last Modified: October 26, 2020
Protocol Integer ID: 43749
The 1% bacteria (PYB1s-yqjF3rd-luxCDABE) were cultured in LB containing 0.1% streptomycin (50 mg/mL) for 24 h with aeration at 37°C to reach a final density of 1 × 108 cells/mL.
Preparation of 2% Alginate solution, 1% calcium-chloride solution, experimental soil:dissolve 2 g Alginate in 100 mL distilled water (2% w/w) and 1g calciumchloride in 100 mL distilled water (1% w/w). The experiment soil was filtrated prior to use.
Sterilize the 2% Alginate solution, 1% calcium-chloride solution, (121°C, 15min), and experimental soil (121°C, 1.5h).
Add 0, 0.25, 0.5 and 1 mg of DNT powder (0, 250, 500 and 1000 mg/kg) to the wells of a 12-well plate.Take 1g of experimental soil and drop it onto the DNT in the 12-well plate on a clean bench.
The engineered bacteria were added into 2% Alginate solution at a 1:9 (v/v) ratio.
Drop 200 µL of 1% (w/w) calcium-chloride solution into the 1 mL alginate-bacterial suspension droplets (the volume ratio between the Alginate mixture and calcium chloride solution was 5:1). Leave the mix for 1 minute.
The mixed Alginate gel was tiled on the soil.