Team:SCUT-China A/Project Demonstrate
Demonstrate
In order to prove our device can work in real world, we chose one to test the metal iron in the water sample.
To verify whether our products can accurately detect the concentration of heavy metal ions in wastewater
of realistic environment or not, we took the water samples from the factory to conduct an experiment.
We took water samples in two spots, an electroplating factory and a pool in an industrial district. For
mainly manufacturing plating products, the electroplating factory has a great deal of effluent which
contains heavy metal ions.
Besides, we noticed that there are at least six electroplating factories in
Jiahewanggang, Guangzhou City , so we visited there and collected water samples. We collected water from
a pool and a stream there and put certain amount mercury into it to simulate the real environment for
the engineered bacteria testing whether the materials in the real environment world effect the work of
them.
E.coil BL21(pSB1C3- rrnb' terminater+MerR+pMerR+SRRz+rrnb terminater)
Water sample (From Jiahewanggang)
Imitation sample (water sample+10-7M Hg2+)
LB medium+Chloramphenicol
2X LB medium+Chloramphenicol
Mercury standard solution
IPTG
X-gal
Z-buffer
β-mercaptoethanol
ddH2O
1. Grow the cells on LB medium+Chloramphenicol overnight.
2. Translate cultures into LB medium+Chloramphenicol in another conical flask.
3. Incubate the cultures for about an hour.
4. Set your instrument to read OD600 and measure OD600 of the cultures.
5. When OD600 reaches 0.4, add IPTG to the cultures.
6. Incubate the cultures for about half an hour.
7. Centrifuge at 4000g, 10mins to collect 50mL cultures
8. Add 25mL 2X LB medium+Chloramphenicol to resuspend the thalli. And add IPTG.
9. Add the 2.5mL cultures into each cuvette and 2.5mL water sample, Imitation sample, Mercury standard solution, ddH2O.
10. Incubate the cultures at 37℃, 220rpm for about half an hour.
11. Centrifuge at 15000g, 15mins to collect supernatant.
12. In 96-well plate，set up a reaction mix at room temperature containing:
X-gal reaction mix
Z-Buffer (included β-mercaptoethanol )
X-gal
supernatant
13. Use our app to measure the concentration of mercury ions.
After we use smart phone application we made to detect its mercury concentration, the result showed that the positive control’s concentration is 0.0160mg/L, when the imitate sample is 0.0158mg/L. The result is close to the real concentration 0.01mg/L. The error may cause by the environmental element.
What’s important is that this devise can work in the reality water sample while the performance is similar to Mercury standard solution. This demonstrate strongly proved that the devices designed by SCUT-China_A can work in the real world and perform a good performance.
