Part:BBa_K4299001

Uric acid promoter

HucO is regulated by HucR. When uric acid is abscent, HucR can bind to hucO making downstream gene silencing.

Added by Sesame_Shenzhen

1.Validating the functionality of our Uric acid sensor   1.1 Finding the functional concentration of our Uric acid detector To ensure that our Uric acid-sensitive promoters, HucR and HucO, can function properly by initiating different protein expression rates, we made bacteria (E.coli BL21)that connected our promoter with a red fluorescent protein.

Figure 1

We grew the engineered in liquid LB growth medium in different samples, each sample containing different concentrations of Uric acid. After growth for 3 hours at 37 degrees, 220 rpm, we observed our results.

Figure 2

The experimental results found that the control group without uric acid had no red fluorescence, and the experimental group with uric acid concentrations of 10^-6 mol/L and 10^-5 mol/L had only a small amount of red fluorescence. When the concentration of uric acid was 10^-4 mol/L, there was obvious fluorescence, and a large amount of red fluorescence was observed at 10^-3 mol/L.

Therefore, the optimal inducing concentration of uric acid is 10^-4 to 10^-3 mol/L. That is, 100—1000 μmol/L   1.2 Finding the expression curve of our promoter   1. Cultivate engineered strains in LB liquid medium and adjust uric acid to different concentrations. N=3

2. Incubate for 3 hours at 37°C, 220 rpm.

3. Transfer to a 96-well plate

4. The microplate reader detects the red fluorescence luminescence value.

Figure 3

The experimental results show that the engineered strain can function at concentrations between 100 to 1000 μmol/L of Uric acid.   1.3 The effect of time on the Promoter We sampled the fluorescent level of the RFP at intervals of 3 hours with the engineered strand in different Uric Acid concentrations.

Figure 4

Time course experiments showed that the fluorescence intensity became quite strong within 4 to 6 hours after uric acid induction and stabilized within 10 to 12 hours.   Experimental method: 

1. Cultivate engineered strains incubated LB liquid medium, and we adjust uric acid to different concentrations. N=3

2. The temperature is 37 degrees Celsius, 220 rpm.

3. Transferred to a 96-well plate

4. The microplate reader detects the red fluorescence luminescence value.

1.4 Other Factors Influencing our promoter   We experimented if our promoter could function properly under varying conditions. We concluded that our promoter works in an environment with a pH of 5.0 to 9.0 and a temperature of 25-50 degrees Celsius.

Figure 5

The experimental results show that the optimal pH is around 7 and the optimal temperature is 40 degrees Celsius for the Uric Acid promoter to function properly. It also functions at pH 5.0 to 9.0 and a temperature of 25-50 degrees Celsius.   Method:

1. Cultivate the engineered strain in LB liquid medium, with a uric acid concentration of 1000 μmol/L. N=3

2. Adjust different temperatures and pH, 220 rpm, and cultivate for different times.

3. Transfer to a 96-well plate

4. The microplate reader detects the red fluorescence luminescence value.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 546
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 282
  Illegal SapI.rc site found at 246