Team:HBUT-China/Results
After detecting the function of the nickel ion channel protein, we determined that this channel protein could be expressed in E. coli, and can transport more nickel ions into cells. Based on this, we envisaged that if this channel protein were to be integrated with the system for detecting nickel ions, BBa_K2652004, more nickel ions would enter the cell, and thus more would combine with NcrB. Therefore, the binding efficiency of NcrB to pncrA would be greatly reduced, resulting in significantly greater expression of LuxCDABE. This would mean we could detect data with higher luminescence intensity, which would improve our detection accuracy.
Based on BBa_K2652004, we added a nickel ion channel protein gene nikABCDE to construct BBa_K2652005. We called its carriers Nickel Hunter 2.0.
The above picture is the design of our genetic circuit. In order to verify our ideas, we conducted the following experiment.
1) Determining the toxicity of nickel ions to Nickel Hunter 2.0
Since the absorption of nickel ions by the cells is increased after the addition of the nickel ion channel protein NikABCDE, and nickel ions are toxic to the cells, we knew we had to test the tolerance of the cells to nickel ions. We induced the Nickel Hunter 2.0 with 0.0005, 0.005, 0.05, 0.5, 5, 50, 500 mmol/L NiCl2 solution. Cultured overnight. Results are as follows.
According to our modeling analysis, this image should have such c1, c2 values, so that when c(Ni2+)>c1, the number of cells is almost 0. When c(Ni2+)<c2, the number of cells remains stable, but this image is seriously inconsistent with the analysis, and even at the end of the last curve, there is a rebound phenomenon. We suspect this was caused by the cultivation time being too short. So, we extended the cultivation time to 16h for the second measurement. Results are as follows.
As you can see from the figure, when c(Ni2+)>5mmol/L, the number of cells is close to 0. When c(Ni2+)<0.5mmol/L, the number of cells remains stable. Therefore, the maximum tolerance of the BBa_K2652005 carriers is 0.5mmol /L.
2) Determine the concentration of nickel ions detected
According to the data from Nickel Hunter 1.0, when the initial bacterial concentration is 0.6-0.8, we get the largest intensity after we use Ni2+ to induce.
We induced the BBa_K2652005 with 0, 10-7, 10-6, 10-5, 10-4, 10-3, 10-2, 10-1 mmol/L NiCl2 solution. After a period of time, the luminescence intensity was measured by a microplate reader.
As shown in the figure, when c(Ni2+)<10-3mmol/L, the luminescence intensity increases with the increase of nickel ion concentrations, so we set the detection range at 10-7~10- 4 mmol/L.
3) Determine the detection time
In order to determine the best test time, we induced the Nickel Hunter 2.0 with 10-4 mmol/L NiCl2 solution, and detected the luminescence intensity and OD600 at 0h, 2h, 4h, 6h, 8h by microplate reader, and plotted the figure of relationship between nickel ions concentration and LI/OD600. As follows.
As shown in the figure, the value of LI/OD600 gradually increases from 0~6h, and tends to be stable at 6~8h, so we set the detection time to 6~8h.
4) Detection of the relationship between nickel ions concentration and luminescence intensity
We induced Nickel Hunter 2.0 with 0, 10-7, 5×10-7, 10-6, 5×10-6, 10-5, 5×10-5, 10-4 mmol/L NiCl2 solution. After culturing 6~8h, the luminescence intensity and OD600 were measured by microplate reader. Draw FI/OD600-(FI/OD600)0 and Ni2+ curves.
5) Comparison of BBa_K2304004 and BBa_K2652005 (Nickel Hunter 2.0) response to nickel ions concentration
As shown in the figure, the luminescence intensity of BBa_K2652005 is significantly higher than that of BBa_K2652004, so after adding the channel protein for nickel ions, the luminescence intensity that we can detect is significantly increased, thus increasing the detection accuracy; Also, our detection range expanded from 10-6~10-4mmol/L to 10-7~10-4mmol/L.
Result
Background
Experiment
