Engineering S uccess
Overview
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 1,2 . 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 1). Our project aims to use probiotics Nissle 1917 to synthesize the enzymes(UAO, allantoinase, and allantoicase) that can digest the uric acid to work as a cure for gout. We constructed specific plasmids pGex-4T-1-UAO (BBa_K5024007) and pGex-4T-1-UAA (BBa_K5024008), and transformed them in to the target probiotics Nissle 1917.
Figure 1. The engineering schematic diagram of the project
ROUND 1 pGex-4T-1-UAO ( BBa_K5024007 )
DESIGN
Two sections are shown as follow:
pET28a-UAO
We firstly choose pET28a as the vectors for the plasmids with target gene sections UAO. We choose pET28a for the His section it involves, which is crucial in creating pGex-UAO plasmids via homologous recombination and purifying the proteins synthesized with Ni-NTA(The Nicole ions in Ni-NTA ensures that the protein synthesized with HIS sequence can attach to it while other proteins cannot, and the target proteins are thus retained during purification). The Obtained target gene sections after this process thus all contain HIS sequence. The product Uric acid oxidase and pET28a vector were digested with NEB restriction enzymes BamHI and XhoI, and the vector fragment was recovered by gel recovery (Figure 2).
Figure 2. The plasmid of pET28-UAO
pGex-4T-1-UAO
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 3.The picture of pGex-4T-1-UAO plasmid.
BUILD
pET28a-UAO
We extract the pET28a vectors from existing bacteria solutions. Primarily using centrifuging, we extract the vectors from the bacteria solutions. The final products are obtained from the remnants in the absorption column. Then, the vectors are linearized by using restrictive enzymes. The electrophoresis is conducted to examine the products. Cyclical vectors move faster during the electrophoresis than the linearized vectors.
By comparing the horizontal relationships between the stripes of target gene sections and the marker, we have concluded that in the same conditions, the pET28a control group moves faster during the electrophoresis, and the pET28a samples move slower during the electrophoresis. The length of the pET28a vector is 5369 bp, and the control group of Figure 4 is correct. Line 1-2 is the band of pET28a cut with BamHI and XhoI. This phenomenon in the figure indicates that the vectors pET28a in the sample groups have been linearized.
Figure 4. The electrophoresis for testing the linearized vector pET28a.
Note ：
Line1- 2: The band of pET28a cut with BamHI and XhoI
Line 3- 4: The band of pET28a cut with BamHI and NotI
Line 5: The band of pET28a cut with SacI and XhoI
The lengths of gene fragments UAO are 981bp. By comparing the horizontal relationships between the stripes of target gene sections and the marker, Figure 5 concluded that the fragment lengths are consistent with the results. Therefore, the figure indicates that target genes of UAO are successfully amplified.
Figure 5. The electrophoresis for the target genes
Next, we connected UAO with pET28a by T4 ligase, and transformed the plasmid into DH5α. Figure 6A 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 6C concluded that the fragment lengths are consistent with the results. We have successfully the vector pET28a-UAO was transformed into DH5α, and the sequencing results in Figure 6B showed no gene mutation. It indicates that our plasmid has been successfully constructed.
Figure 6. The Electrophoresis of the Monoclonal Antibody in DH5
pGEX-4T-1-UAO
The length of UAO is 981 bp. Figure 7A 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 7B). 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 7. 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 8A and B show that the vector pGex-4T-1-UAO was transformed into DH5α, The length of UAO is 981 bp. Figure 8B 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 6 CD showed that UAO has no gene mutation. It indicates that our plasmid pGex-4T-1-UAO has been successfully constructed.
Figure 8. The electrophoresis of the Monoclonal Antibody in DH5
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
TEST
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 9A 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 9B shows that the fragment lengths are consistent with the results. The vector pGex-4T-1-UAO was successfully transformed into BL21(DE3).
Figure 9. 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 10 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.
Table1: Protein concentration of UAO (mg/mL)
|
|
IPTG concentration (mmol/L) |
||||
|
Sample # |
0.050 |
0.250 |
0.500 |
0.800 |
1.000 |
|
1 |
1.016 |
1.320 |
5.101 |
0.783 |
0.817 |
|
2 |
0.583 |
0.992 |
4.764 |
0.652 |
0.814 |
|
3 |
0.656 |
1.402 |
4.759 |
0.625 |
0.831 |
|
Average |
0.752 |
0.991 |
4.875 |
0.687 |
0.821 |
|
SD |
0.232 |
0.217 |
0.196 |
0.085 |
0.009 |
Figure 10. 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 11 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 som e proteins.
Figure11. 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 12 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 12A shows that the fragment lengths are consistent with the results. The vector pGex-4T-1-UAO was successfully transformed into Nissle 1917.
Figure 12. 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 13 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 13. The pGex-4T-1-UAO of SDS-PAGE in Nissle 1917
B.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. We use a linear function to fit. Figure 14 shows that the equation y = 1.1529x + 0.2129 is satisfied, and the correlation coefficient is greater than 0.99.
Table 1.Result and curve for the Standard Curve
|
|
Uric acid concentration (μmol/mL) |
||||||
|
Sample# |
0.25 |
0.125 |
0.0625 |
0.0312 |
0.015625 |
0.007313 |
0 |
|
1 |
0.5086 |
0.3475 |
0.2824 |
0.2529 |
0.2338 |
0.2200 |
0.2110 |
|
2 |
0.5060 |
0.3469 |
0.2824 |
0.2536 |
0.2348 |
0.2199 |
0.2120 |
|
3 |
0.5052 |
0.3485 |
0.2832 |
0.2537 |
0.2353 |
0.2201 |
0.2128 |
|
4 |
0.5032 |
0.3475 |
0.2839 |
0.2536 |
0.2348 |
0.2205 |
0.2119 |
|
5 |
0.5042 |
0.3473 |
0.2843 |
0.2542 |
0.2355 |
0.2203 |
0.2129 |
|
average |
0.50544 |
0.34754 |
0.28324 |
0.2536 |
0.23484 |
0.22016 |
0.21212 |
|
SD |
0.002056 |
0.00059 |
0.000862 |
0.000464 |
0.000658 |
0.000241 |
0.000773 |
Figure 1 4 . 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 2 and Figure 1 5 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.
Table 2. Uric acid oxidase activity of pGEX-4T-1-UAO
|
Concentration of UAO(mg) |
Experimental group |
SD |
Control ( E.coil BL21 ) |
SD |
|
0.00000 |
0.23052 |
0.00058103 |
0.23583 |
0.002199284 |
|
0.01335 |
0.22330 |
0.00095916 |
0.23493 |
0.007408525 |
|
0.02670 |
0.18474 |
0.00102489 |
0.23918 |
0.006044871 |
|
0.04005 |
0.15558 |
0.00051146 |
0.22762 |
0.011768322 |
|
0.05340 |
0.15154 |
0.00127843 |
0.22007 |
0.014096617 |
Figure 1 5 .The curve uric acid oxidase activity of pGEX-4T-1-UAO
LEARN
Analysis of experimental skills:
|
Issue/Phenomenon |
analysis |
solution |
|
The spread of dH5alpha bacteria’s colonies on solid culture plates is not obvious: There aren’t visible spots that refer to dH5alpha bacteria colonies. |
The procedure of preparing and applying the bacteria on the culture plate may involve operational mistakes: The time for shaking bacteria solutions may be inadequate, which results in inadequate amount for the dH5alpha on the plate for observation; the bacteria solution may have been applied violently, which damages the bacteria, thus induces failure for their growth.
|
Extend the bacteria shaking time for 2 hours; Improve the applying operation to ensure that the bacteria are evenly and mildly spread. |
|
According to the results of SDS-PAGE, the stripes referring to target proteins(UAO) are not obvious. However, the stripes referring to the protein supernatant just after ultrasonication are obvious. |
The problem may be induced by two reasons:
|
Using the samples preserved, operate the entire procedure again: Extend the time that the protein supernatant has contacted to the Ni-NTA resin and reduce the volume of the His-A buffer used. |
In future studies, other iGEM teams can optimize the expression levels of urate oxidase (UAO). You will further test the protein expression and enzyme activity of pGEX-4T-1-UAO in Nissle 1917 to find the optimal expression conditions. You also need to validate the ability of urate oxidase (UAO) to degrade uric acid in large quantities. Additionally, The role of urate oxidase in degrading uric acid in the body is still pending verification. The safety of probiotics also requires further validation. Our project can provide data support for the treatment of gout. To establish a new treatment method, we think not only for our country is very practical, but will also have a good application prospect in the world.
ROUND 2 pGex-4T-1-UAA ( BBa_K5024008 )
DESIGN
Two sections are shown as follow:
pET28a-allantoinase and pET28a-allantoinase
We firstly choose pET28a as the vectors for the plasmids with target gene sections allantoinase and allantoicase. We choose pET28a for the HIS section it involves, which is crucial in creating pGex-4T-1-UAA plasmids via homologous recombination and purifying the proteins synthesized with Ni-NTA. The obtained target gene sections after this process thus all contain HIS sequence. The product Uric acid oxidase and pET28a vector were digested with restriction enzymes BamHI, XhoI and NotI, and the vector fragment was recovered by gel recovery (Figure 1 6 ).
Figure 1 6 . The plasmid map of pET28a-allantoinase and pET28a- allantoinase
pGex-4T-1-UAA
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. 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. We use homologous recombination to assemble allantoinase and allantoicase into pGEX-4T-1-UAA(Figure 1 7 ).
Figure 1 7 . The plasmid map of pGEX-4T-1-UAA
BUILD:
pET28a- allantoinase and pET28a- allantoicase
We added restriction sites BamHI and NotI to primer, and further amplified the target gene a llantoinase by PCR. The length of the target gene a llantoinase is 1485 bp. Figure 17 shows that the fragment lengths are consistent with the results. We added restriction sites SacI and XhoI to primer, and further amplified the target gene allantoicase by PCR. The length of the target gene allantoicase is 1288 bp. Figure 1 8 shows that the fragment lengths are consistent with the results. It that we have successfully amplified the target gene allantoicase.
Figure 1 8 . Electrophoresis for the target genes allantoinase and allantoicase
Then, we cut the vector pET28a with restriction endonuclease, and used the uncut vector as a control. Line 1-2 is the band of pET28a cut with BamHI and XhoI in the figure 1 9 . Line 3-4 is the band of pET28a cut with BamHI and NotI. Line 5 is the band of pET28a cut with SacI and XhoI in the figure 18. The results show that the plasmid has been cut into linearization.
Figure 1 9 . The electrophoresis for testing the linearized vector pET28a
Note ：
Line1- 2: The band of pET28a cut with BamHI and XhoI
Line 3- 4: The band of pET28a cut with BamHI and NotI
Line 5: The band of pET28a cut with SacI and xhoI
Next, we connected a llantoinase and allantoicase with pET28a by T4 ligase, and transformed the plasmid into DH5α. Figure 20 A shows that the isolated colonies were successfully grown, and the isolated colonies were selected for colony verification. The length of the target gene a llantoinase is 1485 bp. Figure 20 C shows that the fragment lengths are consistent with the results. The length of the target gene allantoicase is 1288 bp. Figure 20 C shows that the fragment lengths are consistent with the results. We have successfully the vector pET28a-UAO, pET28a- a llantoinase and pET28a- allantoicase were transformed into DH5α, and the sequencing results in Figure 20 B showed no gene mutation. It indicates that our plasmid has been successfully constructed.
Figure 20 . The Electrophoresis of the Monoclonal Antibody in DH5α
Note:
A Culture plates UAO, allantoinase and allantoicase for DH5α
B.Electrophoresis of the Monoclonal Antibody UAO, allantoinase and allantoicase
C.The sequencing result of pET28a-UAO,pET28a- a llantoinase and pET28a-allantoicase
B. pGex-4T-1-UAA
The length of the target gene a llantoinase is 1485 bp. The length of the target gene allantoicase is 1074 bp. Figure 2 1 A shows that the fragment lengths are consistent with the results. The length of the plasmid pGex-4T-1-UAO is 5301 bp. Figure 2 1 B shows that the fragment lengths are consistent with the results. It indicates that we have successfully amplified the target gene.
Figure 2 1 . Electrophoresis for the genes a llantoinase, allantoicase and pGex-4T-1-UAO
Next, we transformed the plasmid pGex-4T-1-UAA into DH5α. Figure 2 2 AB shows that the isolated colonies were successfully grown, and the isolated colonies were selected for colony verification. The length of the target gene a llantoinase is 1485 bp. Figure 2 2 C shows that the fragment lengths are consistent with the results. In Figure 2 2 D, line1,4,5 shows that the allantoicase band is approximately 1024bp. It indicates that we have successfully translated the plasmid pGex-4T-1-UAA to E.coil DH5α.
Figure 2 2 . The electrophoresis of the monoclonal antibody in E.coil DH5α
Afterward, we chose positive isolated colonies 1, 4, and 5 and sent them to the company for sequencing. The results showed that the sequencing of the allantoicase and allantoinase genes on the pGex-4T-1-UAA plasmid was normal (Figure 2 3 ), without any genetic mutations. It indicates that our plasmid pGex-4T-1-UAA has been successfully constructed.
Figure 2 3 . The sequencing of the allantoicase and allantoinase genes on the pGex-4T-1-UAA plasmid
TEST
Protein expression
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-UAA into E.coil BL21(DE3). Figure 2 4 A and B show that the isolated colonies were successfully grown, and the isolated colonies were selected for colony verification. The length of the target gene allantoinase is 1485 bp and allantoinase is 1047bp. Figure 2 4 C and D show that the vector pGex-4T-1-UAA was successfully transformed into E.coil BL21(DE3).
Figure 2 4 . The electrophoresis of the monoclonal antibody for pGex-UAA in BL21(DE3)
Note:
Figure 23A-B: The monoclonal plate pGex-UAA in BL21(DE3)
Figure 23C: The Electrophoresis verification of allantoicase in pGex-UAA (BL21(DE3))
Figure 23d: The Electrophoresis verification of allantoinase in pGex-UAA (BL21(DE3))
In order to improve the expression of UAA protein, we designed different gradients of IPTG induction. The size of the UAA protein is 118.6 kDa. Figure 2 5 shows that the crude protein has the target protein UAA. However, 0.1mmol, 0.25 mmol, 0.5mmol ,0.7mmol and 1mmol IPTG had no significant effect on the expression of UAA protein.
Figure 2 5 . The pGex-4T-1-UAA of SDS-PAGE in E.coil BL21(DE3)
Enzyme activity detection
1.The urate oxidase activity of UAA
In order to determine the ability of our constructed plasmid pGex-4T-1-UAA to decompose uric acid, we used uric acid as a substrate and added UAO protein. Table 3 and Figure 2 6 showed that with the increase of UAA protein concentration, the content of uric acid gradually decreased, indicating that UAA 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.
Table 3. The urate oxidase activity of UAA
|
Concentration of UAA(mg) |
Experimental group |
SD |
Control |
SD |
|
0.00000 |
0.247288 |
0.015616036 |
0.194080567 |
0.089358727 |
|
0.01335 |
0.215044 |
0.020638737 |
0.188096847 |
0.084278254 |
|
0.02670 |
0.186315 |
0.010903707 |
0.182951779 |
0.088550902 |
|
0.04005 |
0.142994 |
0.021953815 |
0.169561617 |
0.081267084 |
|
0.05340 |
0.137083 |
0.018292321 |
0.163117255 |
0.079973725 |
Figure 2 6 . The curve of UAA activity
2.Contrast enzyme activity of UAO and UAA
By comparing the levels of uric acid after adding UAA and UAO proteins(Figure 2 7 ), it was found that the uric acid content was lower with UAA compared to UAO. This suggests that UAA has slightly higher enzymatic activity than UAO, further indicating that UAA has a stronger ability to degrade uric acid. However, the protein expression level of UAA can be optimized in order to enhance its enzymatic activity in subsequent experiments.
Figure 2 7 . The curve of UAA and UAO activity
LEARN
Analysis of experimental skills:
|
Issue/Phenomenon |
analysis |
solution |
|
There is almost no result in the electrophoresis examination for the pGex-UAA plasmids. The stripes are not evident or sheer absent. |
This may be induced by two reasons: 1. the occurrence of hybrid bacterium in the incubated dH5alpha samples. These bacteria aren’t able to replicate the plasmids, or they don’t contain target plasmids. 2. the time for heat shock when transforming the plasmids to the dH5alpha bacteria is inadequate. Which induces results that plasmids are not fully transformed to the dH5alpha. |
Operate the entire procedure again: select appropriate bacteria solutions of dH5alpha and extend the transformation time from 12-16 hours to 24 hours. |
|
One of the sample groups shows the concentration of uric acid inconsistent with the concentrations of the proteins. |
This may be induced by two reasons:
|
Create all the samples for the experiment again and measure the values of absorbance. When creating the mixture, notice the concentration of the uric acid and enzymes. |
In future studies, other iGEM teams can optimize the expression levels of fusion protein (urate oxidase, allantoinase, and allantoicase). You will further test the protein expression and enzyme activity of pGEX-4T-1-UAA in Nissle 1917 to find the optimal expression conditions. You can also validate the ability of pGEX-4T-1-UAA to degrade uric acid in large quantities. Additionally, The role of urate oxidase in degrading uric acid in the body is still pending verification. The safety of probiotics also requires further validation. Our project can provide data support for the treatment of gout. To establish a new treatment method, we think not only for our country is very practical, but will also have a good application prospect in the world.
Reference
1. T. Neogi, Clinical practice. Gout. N Engl J Med 364 , 443-452 (2011).
2. 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).
