Part:BBa_K4301002
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Indentify the working range of uric acid detector
I. Experimental Pupose:
identify the scope of work of the uric acid detector In order to precisely estimate the working range of the uric acid detector, we constructed an engineering strain of uric acid sensor-linked RFP.
II. Experimental Process
1.Engineered strains were grown in LB liquid medium and uric acid was adjusted to different concentrations. N=3
2.Incubate for 3 hours at 220 RPM at 37 ° C.
3.Observed under a fluorescence microscope. RFP (red fluorescent protein) was used as a reporter gene to verify the feasibility of our HucR sequence. RFP was not represented when uric acid was absent or not high; when the uric acid concentration was sufficient, RFP was represented and red fluorescence could be detected.
III. Experimental Results
Through the experiments, we acknowledged the range of optimal uric acid induction concentration.
1.We transformed the plasmid into BL21 strain and carried out relecant experiments.
2.From the experimental results, it was found that without the uric acid, the control group had no red fluorescence, while the experimental group with uric acid concentration of 10^-6 mol/L and 10^-5 mol/L had only a small amount of red fluorescence. At the point of uric acid concentration of 10^-4 mol/L, fluorescence was evident at the beginning, and a large amount of red fluorescence was observed at 10^-3 mol/L.
3.Conclusion: the optimal uric acid induction concentration was 10^-4 to 10^-3 mol/L, namely, the 100-1000 mu mol/L.
Detect the curve of the uric acid detector in the working range
I. Experimental Purpose
To better present the relationship between RFP and uric acid concentration during the experiments, we identified the curve of the uric acid detector in the operating range by detecting the red fluorescence luminescence values.
II. Experimental Process
1.Cultured the engineered strain in LB liquid medium, and adjusted uric acid to different concentrations. N=3
2.Temperature 37 degrees Celsius, 220 RPM, 3 hours.
3.Transfered to a 96-well plate
4.Red fluorescence luminescence value was detected by microplate reader
III. Experimental Results
From the scatter diagram we can see that our engineered strains were able to normally perform work at a concentration that ranged much wider than the control group. Under normal circumstances, an adult with a uric acid level higher than 420μmol/L will be considered hypouricemia. The experimental results showed that the engineered strains were able to normally perform work at concentrations ranging from 100 to 1000μmol/L UA. From this, we can deduce the conclusion that our uric acid detector will be able to effectively detect uric acid.
Verify the effect of time on uric acid detector
I. Experimental Purpose
In order to determine the effect of time on the uric acid sensor, we conducted experiments on the fluorescence brightness of engineered bacteria with time course at the concentrations of 100, 500, and 1000μmol/L UA. N=3 times technical replicates.
II. Experimental Process
1. Cultured the engineered strain in LB liquid medium, and adjusted uric acid to different concentrations. N=3
2. Temperature 37 degrees Celsius, 220 RPM, culture for different times
3. Transfered to a 96-well plate
4. Red fluorescence luminescence value was detected by microplate reader
III. Experimental Results
Time-course experiments proved that the fluorescence intensity became quite strong within 4 to 6 h after UA induction and became stable within 10 to 12 h.
Verify the influence of other environmental factors on uric acid detector
I. Experimental Purpose
In order to verify the effect of environmental factors on the uric acid detector, such as pH and temperature, we also tried to create more conditions to test whether the system could work properly in different environments. For example, we tested the condition of pH 5.0 to 9.0, and at temperature 25 to 50 degrees Celsius.
II. Experimental Process
1. The engineered strain was cultured in LB liquid medium, and the uric acid concentration was adjusted to 1000μmol/L. N=3 2. Adjust different temperature and PH, 220 RPM, cultured for different time intervals 3. Transfered to a 96-well plate 4. Red fluorescence luminescence value was detected by microplate reader
III. Experimental Results
The experimental results demonstrated that the most congenial pH was about 7, the best temperature was 40 degrees Celsius. The uric acid detector also worked at pH 5.0 to 9.0 and at temperatures between 25 and 50 degrees Celsius.
This suggested that the uric acid detector hardware should be stable in an environment with pH value of 7 and temperature of 40℃. However, the uric acid detector hardware was difficult to maintain at 40 degrees Celsius, and experimental results also indicated that it could work well at other temperatures.
Concept Demonstration [Part 2]: amplification system
Validation of the amplification system
I. Experimental Purpose
The natural HRP regulatory network contained the activating proteins HrpR and HrpS. The two would form a hypersensitive high-order co-composite HrpRS. This had been shown to amplify the input transcriptional signal. To verify the magnification of the amplifier, we used promoter LacUV5 as the input to the HRP amplifier and ERFP as the output to complete the verification.
To better illustrate this process, we drew the following diagrams.
We constructed the architecture of transduced signal amplifier based on HRP regulation system.
LacUV5 promoter, genes downstream of which would be transcribed in response to IPTG activation. HrpR and hrpS were the gene encoding the activator protein PhrpL, a promoter induced by hypersensitive high-order co-complex HrpRS. The ribosome binding site (RBS) was B0030, and the terminator was B0015. Red fluorescent protein (RFP) was used as our reporter protein.
II. Experimental Process
In order to induce the expression of the experimental group and the control group, four different concentrations of IPTG (0, 10-6, 10-5, 10-4, 10-3 mol/L) were used in the experiment. After IPTG induction for 1 h, we used an enzyme marker to detect red fluorescence signals with excitation wavelength of 587nm and absorption wavelength of 610nm. N=3
III. Experimental Results
The experimental results shown that the amplification system could transmit about four times the signal.
Concept Demonstration [Part 3]: overall system
The demonstration of overall system
I. Experimental Purpose
Urine containes substances other than uric acid, which might affect the detection system. To confirm this effect, we verified the working efficiency of the whole system, (sense+amplification) including all systems in the simulated urine.
II. Experimental Process
1.Adjusted the concentration of uric acid to different concentrations in the simulated urine. N=3
2.Added the strain (cultivated)
3.Adjusted 37 ° C, PH=7, 220 rpm, and incubate for 3 ° C.
4.Moved to 96 orifice plate
5.The red fluorescent luminescence value was detected by the microplate reader.
III. Experimental Results
The experimental results showed that the system could still work normally in the simulated urine environment. In this way, according to all the experimental results mentioned above, it was demonstrated that our uric acid detector was able to be utilized in actual environment.
Uric acid receptor + cascade amplifier
We used HucR, a transcriptional regulator, to sense uric acid concentrations. HucR, a transcriptional regulator from d. radiodurans strain, belongs to the MarR transcriptional regulator family. This transcription factor binds to a single binding site in the promoter region shared by two genes with high affinity, inhibiting its own expression and the expression of adjacent uricase. The transcription regulator HucR binding site is called hucO. HucO is a common sequence of HucR and urate oxidase promoter in radioresistance cocci. When uric acid is absent, the HucR protein binds to hucO and blocks RNA polymerase binding to prevent expression of the gene involved. In the presence of uric acid, the binding of HucR protein to hucO is inhibited by uric acid molecules; therefore, HucR protein is unable to interact with hucO to activate the expression of related genes. A cascade amplifier consists of two modular terminals -- input and output. Using the hrp operon from Pseudomonas syringae builds. The natural hrp regulatory network contains the activating proteins HrpR and HrpS. These two components will form a hypersensitive higher-order co-complex HrpRS that binds the upstream activator sequence of the hrpL promoter by ATP hydrolysis to remodel the closed σ54-RNAP-hrpL transcription complex into an open one. This has been shown to amplify the output transcriptional signal.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 58
Illegal NheI site found at 81 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 3317
Illegal AgeI site found at 1927
Illegal AgeI site found at 1933
Illegal AgeI site found at 2068
Illegal AgeI site found at 2226
Illegal AgeI site found at 2904
Illegal AgeI site found at 2910
Illegal AgeI site found at 2958 - 1000COMPATIBLE WITH RFC[1000]
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