Part:BBa_K3311004
HucR-pHucO-mCherry
The “HucR-pHucO-mCherry” is composed of “Anderson Strong-RBS-HucR”(BBa_K3311003) and “pHucO-mCherry”(BBa_K3311002 ). It is a important tool for our part function test. HucR is promoted by a constitutive promoter, while the transcription of mCherry is controlled by pHucO.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
As for “HucR-pHucO-mCherry” (Figure 2), while uric acid is absent, HucR will bind to pHucO operator, inhibiting the transcription of downstream genes--mCherry. In this situation, the color of the sample will not change. While uric acid is present, it will change the structure of HucR, causing it to leave pHucO, allowing mCherry to transcript, the sample will turn red. Now, we successfully constructed the plasmid “HucR-pHucO-mCherry”（Figure1-2）.
Added by LZU-HS-Pro-B
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
The experimental results showed that the engineered strains were able to normally perform work at concentrations ranging from 100 to 1000μmol/L UA.
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.
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