First, we constructed a basic sensor and response system to reflect the level of uric acid using red light emitting from the red fluorescence protein. HucR, a transcriptional regulator found in the MarR family from the Deinococcus radiodurans strain, has the ability to bind the only binding site on promoter hucO. Theoretically, the presence of HucR can inhibit the translation of this gene and thus prevent its protein to be synthesized. However, Uric Acid acts as a substrate for HucR. Once HucR bind with uric acid, Phuco's transcription process is no longer obstructed and thus can transcribe its gene and transcribe protein on the subsequent RBS( ribosome binding site) to produce red fluorescence protein needed for signal detection. 

We obtained the gene fragments we designed through DNA synthesis.

-First, the plasmid was extracted for PCR amplification. Then double digestion of PCR fragments and plasmids, nucleic acid electrophoresis, and gel recovery experiment were carried out.

-The PCR fragments and plasmids digested successfully were recovered. The target gene and plasmid were linked to transforming the linked product.

-After the screening, the plasmid was successfully constructed.

-Prepare receptor cells, and transform the engineering vector into the expression host E.coli BL21.

We need to verify the feasibility of our HucR sequence as the sensor system and red fluorescent protein (RFP) as a reporter gene. We also need to test the precision and accuracy of red fluorescent protein (RFP) when discriminating different concentrations of uric acid. We had done a series of experiments to examine the function of our design under different environmental conditions.

In the preparation phase, we engineered strains of BL21 bacteria strain by transferring the plasmids into them. Next, under the condition of a temperature of 37 degrees Celsius and 220 revolutions per minute, incubation was carried out for 3 hours. We will use the data from this group of different concentrations of uric acid as our control group. We then test the data for red fluorescent protein (RFP) at different times, temperatures, and pH.

Using an excitation light irradiation device to detect the fluorescence intensities of different uric acid levels, We also observed the corresponding light intensity of RFP under different uric acid concentrations.  We noticed that RFP is only slightly expressed when uric acid concentration is above 10^-4 mol/L. The majority of visible luminescence occurs only when we increase the uric acid concentration to 10^-3 mol/L. Conclusion: Our system is most effective under the uric acid concentrations of 10^-3mol/L to 10^-4 mol/L. 

To have a more concrete understanding of how uric acid directly influences the light intensity of the RFP protein, we examined the relationship between light intensity of pH levels to test out the range of uric acid concentration of system can tolerate. 

Under the same environmental circumstance as the previous group( 37 degrees Celsius and 220 revolutions per minute, incubation carried out for 3 hours), we controlled the change in light intensity as compared to the control group, the bacteria strain without any engineering. Under observation, we noticed a strong positive correlation between uric acid concentration and light intensity from our engineered bacteria strain. The light intensity grew as expected as we continued to increase our uric acid level. As we continued to increase uric acid concentration, we reached the limit of 1000μ mol/L where light intensity reached maximum illumination. In conclusion, our system functions under uric acid concentrations from 100-1000μmol/L.

We also tested out the time interval needed for RFP to be fully expressed Under the same environmental conditions(37 degrees Celsius and 220 revolutions per minute, incubation carried out for 3 hours), and during three trials of 100、500、1000μmol/L uric acid concentration, we tested out the time our system needed to maximize their intensity and when does it stabilize. 

An increase in light intensity of the RFP protein is more significant when uric acid concentration is higher, as in 1000μmol/L and 500μmol/L. Changes in 100μmol/L were less apparent so we relied on comparison and evaluation of the general pattern exhibited in the three groups. We, at last, noticed that on average, uric acid-induced luminosity reaches its maximum after 4-6 hours and stabilizes after 10-12 hours. 

Aside from normalized environmental circumstances, we are cautious about aberrants in real-life situations when our product is applied. Therefore, we tried out different temperatures and pH levels during the incubation period under a stable uric acid concentration of 1000μmol/L. Under temperature range from 10-50 Celcius degrees and 6.0 to 8.0 pH values, we noticed that light intensity is highest at 40 Celcius and ph value 7.0. Though maintaining a temperature as high as 40 Celcius in a real-world setting can be challenging, our system can function at a temperature range of 20-50 Celcius, only with less protein expression. 

When analyzing the outcomes of our experiment， we noticed that the sensitivity of the mere sense system and the luminous system is too low. It was found that the signal was hardly large enough for the actual application. Therefore, we also need to devise another way to amplify the signal of uric acid and render larger results to be detected.

We noticed the importance of enhancing the visibility of our system, ie, amplifying the signals released by RFP protein. The natural HRP regulatory network contained the activating proteins HrpR and HrpS. The two would form a hypersensitive high-order co-composite HrpRS. This has been shown to amplify the input transcriptional signal.

  In this way, we considered the architecture of transduced signal amplifier based on the HRP regulation system in order to achieve this demand. Promoter LacUV5, genes downstream of which will be transcribed in response to IPTG activation. HrpR and hrpS are the genes encoding the activator protein PhrpL, a promoter induced by hypersensitive high-order co-complex HrpRS. The ribosome binding site was B0030, and the terminator was B0015. Red fluorescent protein was used as our reporter protein.

We obtained the gene fragments we designed through DNA synthesis.

-First, the plasmid was extracted for PCR amplification. Then double digestion of PCR fragments and plasmids, nucleic acid electrophoresis, and gel recovery experiment were carried out.

-The PCR fragments and plasmids digested successfully were recovered. The target gene and plasmid were linked to transforming the linked product.

-After the screening, the plasmid was successfully constructed.

-Prepare receptor cells, and transform the engineering vector into the expression host E.coli BL21.

For the experimental results, it was evidence presented that the perceptive rate of uric acid greatly increased after we added the amplification system into our experiment. We have discovered and concluded the general rule that the amplification system we designed and added was able to amplify the signal by a factor of four, which enabled us to better sense and detect the amount of uric acid in our bodies.