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Team:Gaston Day School/cadmiumdetector
From 2014.igem.org
Welcome to the 2014 GDS iGEM Wiki
Cadmium Detector Project Overview
The surrounding areas of Duke Energy’s Buck Steam Station have unintentionally been affected with millions of tons of coal ash containing multiple toxic chemicals including Cadmium. The release of this ash has caused the water to become a health hazard with the potential to cause a wide range of symptoms: flu-like symptoms, kidney damage, fragile bones, and possibly death through prolonged exposure. To minimize the damage caused by Cadmium in water both locally and globally, our 2012 team created several heavy metal detectors, but in 2013 we decided to concentrate on the Cadmium detector. The detector responds to the presence of Cadmium with green fluorescence. In 2014, the team worked to increase the sensitivity levels of our detector. Our detector needed to be able to respond to Cadmium at low enough levels that the detection would be useful and the presence of cadmium would not already be apparent. This year, we completed the addition of the 2007 Cambridge team's sensitivity tuners to our detector. The sensitivity tuners amplify the signal received by the detector. At the end of last year, after adding the sensitivity tuners, we began to see indications of a peak at lower levels of Cadmium than we had previously thought. To define that peak, we used test points that were closer together. We discovered a peak of fluorescence and identified our detection points.
Cadmium Detector Construction
Methods
Starting Sensitivity Tests
Determine which samples you will run
Find both the positive and negative control samples
Obtain 6 tubes for each sample
Add 4 mL of LB (and appropriate antibiotic for the sample)
Add the following amounts of 1 M Cadmium Chloride to the respective tube
1 = 0 mM = 0 ul
2 = 1 mM = 4.1 ul
3 = 5 mM = 20.5 ul
4 = 10 mM = 41 ul
5 = 50 mM = 205 ul
6 = .1 M = 410 ul
Add 100ul of the appropriate cells to each tube
Mix well by inverting / vortexing
Grow overnight
Reading Sensitivity Results
Spin cells down for 5’ in clinical centrifuge
Decant supernatant
Resuspend in 1 mL of 1x PBS
Open Logger Pro
Plug in SpectroVis
Fill one cuvette with PBS
Click “Experiment” “Calibrate”Spec. 1
DO NOT SKIP WARMUP
Place “0” in SpectroVis
Click “Finish Calibration” ”Okay”
Click on the rainbow mountain
Click “Absorbance vs. Concentration”
Select 600.9 nm
Click “Okay”
Take an OD 600 reading for each sample
CHANGE TO FLUORESCENCE
Experiment Change Units Spec. 1 Flu. 405
Click rainbow mountain
Change wavelength to 380.1 nm
Measure a fluorescence reading for each sample
If the reading is above 1:
Take 100 ul of the sample and place in a clean cuvette
Add 900 ul of 1x PBS
Repeat until reading is below 1
Record the number of dilutions
Calculate the “new” flu. reading
Divide the fluorescence reading by its OD 600 reading and record answer as Flu. / OD
Graph and analyze
Cadmium Detector Results
