Part:BBa_K3007012

Humanized mammalian urate-dependent transsilencer (mUTS)

Mammalian urate-dependent transsilencer (mUTS) expresses a fusion protein which contains human KRAB (Krueppel-associated box) domain and a hypothetical uricase regulator HucR domain. HucR works as a homo-dimer and binds to hucO in the absence of UA with high affinity [1]. The KRAB domain could enhance the inhibition efficiency when HucR-hucO interaction exists [2]. Only when the UA level is high enough, HucR will release from hucO, and the downstream gene expresses.

Contribution

Group: QHFZ-China iGEM 2019
Author: Cheng Li
Design:

Figure 1. Schematic cartoon of the DNA construct of BBa_K3007012

Documentation:
Fig. 1 shows our design of the UA removal system. mUTS constitutively expresses and binds to hucO8 module in the absence of UA, which inhibits the expression of smUOX. When the UA level is high, the small molecules can be transported into cytoplasm by URAT1. High-level UA makes HucR release from hucO8, therefore smUOX expresses and degrades UA to allantoin. Here are our demonstration experiments.

Figure 1. Gene circuits designed for uric acid removal. (A) Schematic diagram of main parts. (B) Schematic diagram of the mechanism of the gene circuit.

Firstly, we tested if we could measure UA concentration in samples quantitatively. We prepared a series of standard UA solution sample and measured them by the uric acid detection kit (Nanjing Jiancheng Bioengineering Institute, C012-2). Fig. 2A showed the results which were linearly related to the UA concentration. It indicated we could estimate the UA concentration in the sample based on the absorbance in the following experiments.
Next, we introduced one plasmid (expressing hucO8-smUOX) or two plasmids (expressing URAT1 and hucO8-smUOX, respectively) into human HeLa cells, while using eGFP as a negative control, which was placed on plasmid pEGFP-N1. Because there was no mUTS in HeLa cell line, we considered the expression of smUOX was constitutive. The total amount of plasmids in every group was balanced by pEGFP-N1. After transfection, cells were cultured in different initial concentrations of UA. We measured remaining UA concentration of each sample, and found that smUOX could degrade UA successfully, while URAT1 could enhance the clearance efficiency of smUOX (Fig. 2B). After 2 days culture, the cellular morphology kept stable in all cells (Fig. 2C), which meant HeLa cells could grow normally even in high-level UA addition. These results indicated that URAT1 and smUOX could transport and degrade UA in HeLa cells.

Figure 2. Function test of smUOX and URAT1. (A) A standard curve of the relationship between UA concentration in cell culture medium and OD510 after being mixed with uric acid detection reagent. Data was shown as mean ± SD. N = 3 technical repetitions. (B) Remaining UA concentration of samples from 0 to 48 h. The time when UA was added was defined as 0 h. UA concentration was converted from the absorbance data by the linear correlation equation shown in Fig. 2A. “No cell” group was blank control. Data were shown as Mean ± SD. For “No cell” and “GFP” groups, N = 2 biological repetitions; For the other groups, N = 3 biological repetitions. NS, not significant, P ≥ 0.05; **, P < 0.01; ***, P < 0.001. (C) Photos of different HeLa cells groups. BF, bright field.

Then we wondered that if the mUTS-hucO8 system could control the expression of downstream gene according to extracellular UA concentration. Using hucO8-eGFP as a reporter, we tested the system directly. We transfected 0.3 μg plasmid containing mUTS per well to experimental groups. After 2 days culture, the cells in low UA level didn’t get green, which indicated the mUTS showed inhibition function. However, comparing with the positive control, even if the concentration of UA was 800 μM, the production of eGFP was still rare (Fig. 3A). That meant the interaction of mUTS to hucO8 was over strong, even high-level UA could not release the inhibitor from DNA. A hypothesis was the expression of mUTS was too much.
To optimize behavior of our UA removal system, changing promoter into a weaker one was a straightforward way. However, the promoters of eukaryotes are more complex than prokaryotes. As a result, we reduced the amount of plasmid expressing mUTS during transfection, from 0.3 μg / well to 0.1 μg / well. In this situation, hucO8-eGFP could express under the induction of high-level UA (Fig. 3B). The quantitative result indicated mUTS-hucO8 system worked as we predicted: if UA concentration is low, mUTS powerfully suppress the eGFP expression, while under the induction of high UA concentration, the inhibition would be resumed (Fig. 3C, 3D).

Figure 3. Function test of mUTS-hucO8 system. (A) Photos of the cells which were transfected with 0.3 μg / well mUTS expression plasmids at 48 h after uric acid addition. (B) Photos of the cells which were transfected with 0.1 μg / well mUTS expression plasmids at 48 h after uric acid addition. (C) Fluorescence intensity distribution of the cells measured by flow cytometry. NTC, negative control. Population 1 (P1) represented cells that did not successfully intake hucO8-GFP plasmid. (D) Geo. mean of the fluorescence intensity of P2 in Fig. 3C. Data were shown as mean ± SD. N = 3 technical repetitions.

At last, using the optimized system, we changed the hucO8-eGFP plasmid to hucO8-smUOX, and tested the clearance efficiency of UA by our final system. Excitingly, transfection of the combination of URAT1, mUTS and hucO8-smUOX into one cell could reduce the UA concentration in cell culture medium (Fig. 4). What’s more, as the initial UA concentration increased, the clearance rate to UA of our final system became more and more similar to the positive control group, which cells were only transfected hucO8-smUOX.
All the results above indicated that at low UA concentration (such as 400 μM, which was an acceptable concentration for human body), mUTS in UA removal system suppressed the function of smUOX, while at high UA concentration (such as 1600 μM, which was much higher than the standard for human body), smUOX would express and clear uric acid.

Figure 4. Final performance of UA removal system under treatment with different initial concentration of UA. (A) Measurement of the remaining UA concentration in cell culture medium from 0 to 48 h. The initial UA concentrations were 400, 800 and 1600 μM, respectively. The time when uric acid was added was defined as 0 h. The absorbance data was converted into UA concentration by linear correlation equation shown in Fig. 2A. Data were shown as Mean ± SD. N = 3 biological repetitions. (B) Comparation of clearance efficiency between the final system and the positive control. Percentage number referred to the ratio of reduced UA by the final system to the one by positive control. Data were shown as Mean ± SD. N = 3 biological repetitions.

These data support our smart cells could detect and degrade UA molecules in the culture medium. However, the efficiency of UA clearance is not high. There might be several reasons. On the one hand, it may be related to the strong inhibition of downstream genes by mUTS. On the other hand, the transient transfection efficiency of plasmids was not 100%, which indicated not all cells containing the system. In addition, all experiments above were achieved by transient transfection of plasmids. So, we do not know whether the UA removal system could work for a l ong time, or whether the UA concentration in patients’ body fluid can be controlled within a certain range. In the future, we will further optimize the system, including to construct a stable cell lines, to improve the genetic circuit, to demonstrate a long-term cell culture and so on.

References:
[1] Liang, C., Xiong, D., Zhang, Y., Mu, S., & Tang, S. Y. (2015). Development of a novel uric-acid-responsive regulatory system in Escherichia coli. Applied microbiology and biotechnology, 99(5), 2267-2275.
[2] Urrutia, R. (2003). KRAB-containing zinc-finger repressor proteins. Genome biology, 4(10), 231.

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 508
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 244
  Illegal SapI.rc site found at 208