Part:BBa_K5024007
pGEX-4T-1-UAO
pGEX-4T-1-UAO
BBa_K5024007(pGEX-4T-1-UAO)
Construction Design
BBa_K5024007(pGEX-4T-1-UAO) is composed of BBa_K5024006 (pGEX-4T-1) and BBa_K5024000 (Uric acid oxidase). To convert to Nissle1917, we replaced pET28a with pGEX-4T-1. The pGex-4T-1 is chosen for its property of functioning stably in Nissle 1917. The next step is to construct the plasmids that are directly used for target proteins synthesis in following processes. The vectors are pGex-4T-1 and the target genes are HIS-UAO acquired. At the same time, the GST tag of pGEX4T-1 can be replaced with His. We replaced the GST tag on pGEX-4T-1 with His by homologous recombination.
Figure 1. The picture of pGex-4T-1-UAO plasmid.
Engineering Principle
Uric acid is a waste product of the metabolism of purines in the food, most of the uric acid is excreted through liver. However, if the body is unable to efficiently excrete uric acid, uric acid level will rise, forming urate crystal and deposit in the joints and surrounding soft tissue, causing a painful and inflammatory response that leads to joint pain, and swelling. Some animals use urate oxidase to catalyze the conversion of uric acid into allantoin, which helps reduce the accumulation of uric acid in the body.
Figure 2. The engineering schematic diagram of the project
Cultivation, Purification and SDS-PAGE
A. Construction of pGEX-4T-1-UAO plasmid
The length of UAO is 981 bp. Figure 3A shows that the fragment lengths are consistent with the results. It indicates that we have successfully amplified the target gene UAO. We use inverse-PCR to replicate the samples of pGex-4T-1. Inverse-PCR will replicate the section except the section selected. Through inverse PCR, vast amount of incomplete pGex-4T-1 can be perceived. After replication,the results are tested by electrophoresis. The length of vector pGex-4T-1 extracted is 4328bp (Figure 3B). By comparing the horizontal relationships between the stripes of extracted vector and the marker, we have concluded that the vector lengths are consistent with the results. Therefore, the figure indicates that vectors pGex-4T-1 are successfully linearized.
Figure 3. The Electrophoresis for the target genes UAO and pGEx-4T-1 vectors
After constructing the target plasmids pGex-4T-1-UAO, they are transferred to the E.coli DH5α for replication. The figure 4A and B show that the vector pGex-4T-1-UAO was transformed into DH5α, The length of UAO is 981 bp. Figure 4B shows that the fragment lengths are consistent with the results. It proves that the plasmid pGex-4T-1-UAO was successfully constructed. And the sequencing results of pGex-4T-1-UAO in Figure 4 CD showed that UAO has no gene mutation. It indicates that our plasmid pGex-4T-1-UAO has been successfully constructed.
Figure 4. The electrophoresis of the Monoclonal Antibody in DH5α
Note:
A: Culture plates pGex-4T-1-UAO for DH5α
B: Electrophoresis of the monoclonal Antibody pGex-4T-1- UAO
C and D: The Sequencing results of pGex-4T-1-UAO
B. Protein expression
1. pGex-4T-1-UAO (BL21(DE3))
In order to improve the expression of the protein, we first tested the protein in E.coil BL21(DE3). So we transformed the plasmid pGex-4T-1-UAO into BL21(DE3). Figure 5A shows that the isolated colonies were successfully grown, and the isolated colonies were selected for colony verification. The length of UAO is 981 bp. Figure 5B shows that the fragment lengths are consistent with the results. The vector pGex-4T-1-UAO was successfully transformed into BL21(DE3).
Figure 5. The Electrophoresis of the monoclonal antibody pGEX-UAO in BL21(DE3)
In order to improve the expression of UAO protein, we designed different gradients of IPTG induction. Table 1 and Figure 6 showed that the protein concentration of pGEX-UAO increased first and then decreased with the increase of IPTG concentration. When the concentration of IPTG was 0.5 mmol, the protein concentration of UAO was the highest.
Figure 6. The relationship between the concentration of IPTG and proteins
Next, we induced the protein UAO with 0.5 mmol IPTG. The size of the UAO protein is 40 kDa. Figure 7 shows that the crude protein has the target protein UAO, and the purified protein solution also has a small amount of UAO, but the purification loses some proteins.
Figure 7. The pGex-4T-1-UAO of SDS-PAGE in E.coil BL21(DE3)
2. pGex-4T-1-UAO (Nissle 1917)
We transformed the plasmid pGex-4T-1-UAO into Nissle 1917. Figure 8 B shows that the isolated colonies were successfully grown, and the isolated colonies were selected for colony verification. The length of the target gene UAO is 981 bp. Figure 8A shows that the fragment lengths are consistent with the results. The vector pGex-4T-1-UAO was successfully transformed into Nissle 1917.
Figure 8. Electrophoresis of the monoclonal antibody for pGex-UAO in Nissle 1917
Meanwhile, we induced the protein UAO with 0.5 mmol IPTG in Nissle 1917. The size of the UAO protein is 40 kDa. Figure 9 shows that the crude protein has the target protein UAO. Nissle 1917 had no target protein of UAO in the control group. It proved that The protein of UAO was successfully expressed.
Figure 9. The pGex-4T-1-UAO of SDS-PAGE in Nissle 1917
Characterization/Measurement
A. The enzyme activity detection of UAO
1. The Standard Curve
Firstly, we made a standard curve for the determination of uric acid. The table 1 is the original data of the table(Table 2). We use a linear function to fit. Figure 10 shows that the equation y = 1.1529x + 0.2129 is satisfied, and the correlation coefficient is greater than 0.99.
Figure 10. The Standard Curve of Urate Oxidase
2. The enzyme activity of UAO
In order to determine the ability of our constructed plasmid pGex-4T-1-UAO to decompose uric acid, we used uric acid as a substrate and added UAO protein. Table 3 and Figure 11 showed that with the increase of UAO protein concentration, the content of uric acid gradually decreased, indicating that UAO is active and can successfully decompose uric acid. And we used E.coil BL21 (DE3) as the control group, the uric acid content of the control group had no obvious change trend.
Figure 11. The curve uric acid oxidase activity of pGEX-4T-1-UAO
Reference
- T. Neogi, Clinical practice. Gout. N Engl J Med 364, 443-452 (2011).
- Y. Zhu, B. J. Pandya, H. K. Choi, Prevalence of gout and hyperuricemia in the US general population: the National Health and Nutrition Examination Survey 2007-2008. Arthritis Rheum 63, 3136-3141 (2011).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 956
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 930
Illegal XhoI site found at 951 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2089
Illegal SapI.rc site found at 342
Illegal SapI.rc site found at 3171
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