Team:HBUT-China/Inspiration
Background
Nickel Hunter 2.0 is designed to detect nickel ions in the environment. The ABC family proteins expressed by the nikABCDE gene, act as channel proteins. They can transport nickel ions from the environment into the cells, and have a strong transport ability, so that more nickel ions can enter the cells, thereby improving the accuracy of nickel ion detection. We independently verified whether the nikABCDE gene could successfully express the proteins, and how effective it would be transporting nickel ions.
Purpose
1. Through a separate analysis of the nikABCDE gene, we wanted to determine that the gene could express proteins successfully.
2. To measure how much the ability of transporting nickel ions would be improved by the proteins expressed by the nikABCDE gene.
Method
Through Tris-Glycine SDS polyacrylamide gel electrophoresis, we investigated whether the nikABCDE gene would express proteins. Based on the principle that dimethylglyoxime can react with nickel ions to form a complex, we can measure the absorbance of complexes formed by different concentrations of nickel ions and dimethylglyoxime. Thus we obtained a standard curve of the complex and nickel ion concentration.Learn more
After breaking the pSB1C3-01 and pSB1C3-01-nik cells, we then extracted the nickel ions in the cells, and were able to determine the concentration of nickel ions in the cells by measuring the absorbance of nickel ions.Learn more
Results
Discussion
After our experiments with the nikABCDE gene, we found that it could successfully express the proteins, which can transport nickel ions from the environment, into the cells, and showed a high efficiency of transport after measurement. Therefore, the detection accuracy of Nickel Hunter2.0 was greatly improved by applying this system.
data analysis:
1. Protein gel analysis: It could be seen from the picture of the protein gel that in the case of the same Marker standard, the E. coli with nikABCDE is darker than the E. coli without nikABCDE, indicating that there were more proteins expressed by E. coli with nikABCDE. By comparing the length of the ABC protein expressed by the nikABCDE system with the Marker in the protein gel, it could be concluded that the nikABCDE system will express the transport protein which we need in E. coli.
I IV : Marker II :Without nikABCDE gene III :With nikABCDE gene Histogram analysis:
Through the nickel ion concentration standard curve we drew, we could measure the concentration of nickel ions in the cells. By comparison, we could tell that in the E. coli with the nikABCDE gene, the concentration of nickel ions in the cells was significantly higher than that without the nikABCDE gene. And by this comparison, we could conclude that the proteins expressed by the nikABCDE gene increased the efficiency of nickel ion transport by 41%.
Standard curve measurement method: Take 0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 mL of nickel ion solution separately in 10 mL test tubes. Add HCl to 5 mL, and then add 0.5 mL of 500 g/L seignette salt, 1.5 mL of 100 g/L NaOH, and 0.5 mL of 160 g/L ammonium persulphate. Mix the solution and let it stand for 3 minutes. Then add 0.2 mL of 10 g/L dimethylglyoxime-ethanol. Finally, add water to 10 mL. After mixing, let it stand for 15 minutes and measure the absorbance at 470 nm. （Collect 100 mL of the bacterial liquid separately, after centrifuging, discard the liquid supernatant and wash the cells twice with double distilled water and EDTA, and then disrupt the cells using ultrasonication. After that, extract the nickel ions with HCl, and use the filtered solution for measuring the absorbance.）
Inspiration
