Overview
During our experiment, we added some new parts for iGEM part (Table 1), for instance, pET28a-UAO(BBa_K5024003), pET28a-allantoinase (BBa_K5024004 ), pET28a-allantoicase (BBa_K5024005 ), pGEX-4T-1-UAO (BBa_K5024007 ) and pGEX-4T-1-UAA (BBa_K5024008 ). And we also provided some experimental data for reference. The data collected are universally applicable to other related experiments in the field of gene editing. These indices may be effectivein the further investigations that involve protein synthesis and verification, target genes replication, and uric acid digestion. All the results that may be contributable have been listed below.
Table 1. Part contributions
|
Part number |
Part name |
Contribution type |
|
pet28a-backbone |
Add new experimental data and created three new parts, BBa_K5024003 , BBa_K5024004 , and BBa_K5024005 |
|
|
Uric acid oxidase |
Create A New part |
|
|
allantoinase |
Create A New part |
|
|
allantoicase |
Create A New part |
|
|
pET28a-UAO |
Create A New part based on the existing part BBa_K3521004 |
|
|
pET28a-allantoinase |
Create A New part based on the existing part BBa_K3521004 |
|
|
pET28a-allantoicase |
Create A New part based on the existing part BBa_K3521004 |
|
|
pGEX-4T-1-UAO |
Create A New part |
|
|
pGEX-4T-1-UAA |
Create A New part |
Part Contributions
Add new experimental data to part BBa_K3521004 and created three new parts, BBa_K5024003 , BBa_K5024004 , and BBa_K5024005
Summary:
In order to amplify our designed plasmids before we transferred to the Nissle 1917, we firstly connected our target coding genes with pET28a vector ( BBa_K3521004 ) and conducted the amplification in E. coli DH5-alpha host.
pET28a-UAO(BBa_K5024003)
Length: 6253bp
Function: Participates in the decomposition of uric acid, converting Allantoic acid into urea.
Illustration:
Figure 1.1
pET28a- Allantoinase (BBa_K5024004)
Length: 6721bp
Function: Participates in the decomposition of uric acid, converting Allantoic acid into urea.
Illustration:
Figure 1.2
pET28a- Allantoicase (BBa_K5024005)
Length: 6372bp
Function: Participates in the decomposition of uric acid, converting Allantoic acid into urea.
Illustration:
Figure 1.3
Plasmid Construction
W e cut the vector pET28a with restriction endonuclease, and used the uncut vector as a control. The length of the pET28a vector is 5369 bp, and the control group of Figure 1.4 is correct. Line 1-2 is the band of pET28a cut with BamHI and XhoI. 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. The results show that the plasmid has been cut into linearization.
Figure 1.4 . 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
W e connect UAO, a llantoinase and allantoicase with pET28a by T4 ligase, and transformed the plasmid into DH5α. Figure 1.5 A 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. The length of the target gene a llantoinase is 1485 bp. The length of the target gene allantoicase is 1288 bp. Figure 1.5 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 1.5 B showed no gene mutation. It indicates that our plasmid has been successfully constructed
Figure 1.5 . 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
● Add New Part s
The result of electrophoresis for the replicated samples of UAO, allantoinase, and allantoicase.
Analysis: the lengths of gene fragments of UAO, allantoinase, and allantoicase are 981bp, 1485bp, and 1228bp respectively. By comparing the horizontal relationships between the stripes of target gene sections and the marker, we have concluded that the fragment lengths are consistent with the results. Therefore, Figure 1 indicates that three target genes——UAO, allantoinase, and allantoicase——are successfully amplified and are thus able to be used for further procedures about enzyme digestion.
Figure 2. 1. The electrophoresis for target genes(UAO, allantoinase, allantoicase)
Electrophoresis for the target genes (UAO) and the pGEx-4T-1 vectors linearized
Analysis: The length of gene UAO is 981bp respectively. By comparing the horizontal relationships between the stripes of target gene sections and the marker, we have concluded that the fragment lengths are consistent with the results. Therefore, Figure 4 indicates that target gene UAO is successfully amplified and is thus able to be used for further procedures about constructing pGex-4T-1 plasmids.
Analysis: The length of vector pGex-4T-1 extracted is 4328bp. 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, Figure 2.2 indicates that vectors pGex-4T-1 are successfully linearized and are thus able to be used for further procedures about constructing pGex-4T-1 plasmids.
Figure 2.2 . The Electrophoresis for the target genes UAO and pGEx-4T-1 vectors
Culture plates for DH5α E.coli transformed with pGex-UAO plasmids
Analysis: this process indicates the result of transforming the target vectors to the DH5α E.coli and its incubation. When constructing the culture mediums, we have involved Amp+ (Inhibitor of protein synthesis). The observable spots on the culture mediums in Figure 5 indicate that the DH5α E.coli has grown successfully, and they are thus usable in the following procedures of extracting and testing plasmids pGex-UAO.
Analysis: the lengths of plasmids pGex-UAO are 987bp. By comparing the horizontal relationships between the stripes of plasmids and the marker, we have concluded that the fragment lengths are consistent with the results. Therefore, Figure 2.3 indicates that replicated plasmids pGex-UAO are successfully amplified and are thus able to be used for further procedures about transforming into E.coli DH5α. It indicates that our plasmid pGex-4T-1-UAO has been successfully constructed.
Figure 2.3 . The electrophoresis of the monoclonal antibody of pGEX-4T-1-UAO in E.coil DH5α
Protein expression and purification
pGex-UAO plasmids( E.coli BL21)
1.1 pGex-UAO plasmid transform
Analysis: this process indicates the result of transforming the target vectors to the BL21 E.coli (specialized E.coli for protein synthesis) and its incubation. The observable spots on the culture mediums in Figure 2.4 indicate that the BL21 E.coli has grown successfully, and they are thus usable in the following procedures of protein synthesis.
Analysis: the lengths of plasmids pGex-UAO are 987bp. By comparing the horizontal relationships between the stripes of plasmids and the marker, we have concluded that the fragment lengths are consistent with the results. Therefore, Figure 6 indicates that replicated plasmids pGex-UAO are successfully transformed and are thus able to be used for further procedures about protein synthesis.
Figure 2.4 . The Electrophoresis of the monoclonal antibody pGEX-UAO in E.coil BL21(DE3)
1.2 IPTG Induce pGex-UAO ( E.coli BL21)
Analysis: to discover the optimum concentration of IPTG used for induction, the relationship between the concentration of IPTG used and the concentration of proteins synthesized in the sample(Table 1). The following procedures will use the optimum concentration deduced by the curve in figure 2.5 .
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 2.5 . The relationship between the concentration of IPTG and proteins
1.3 SDS-PAGE for the target proteins synthesized and purified(UAO)
Analysis: The feature of the proteins obtained(UAO) is approximately 40 kDa. By comparing the horizontal relationships between the stripes of proteins and the 15% Tris-Glycine marker on figure 8, we have concluded that the target proteins(UAO) are successfully acquired. The samples can thus be used for following processes about enzymatic activity verification.
Figure 2.6 . The pGex-4T-1-UAO of SDS-PAGE in E.coil BL21(DE3)
pGex-4T-1-UAO (Nissle 1917)
Analysis: We transformed the plasmid pGex-4T-1-UAO into Nissle 1917. Figure 2.7 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 9A shows that the fragment lengths are consistent with the results. We have successfully the vector pGex-4T-1-UAO was transformed into Nissle 1917.
Figure 2.7 . Electrophoresis of the monoclonal antibody for pGex-UAO in Nissle 1917
Analysis: we induced the protein UAO with 0.5 mmol IPTG in Nissle 1917. The size of the UAO protein is 40 kDa. Figure 2.8 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 2.8 . The pGex-4T-1-UAO of SDS-PAGE in Nissle 1917
D.Test
1. The Standard Curve
Analysis: we made a standard curve for the determination of uric acid. The table 2 is the original data of the table. We use a linear function to fit. Figure 2.9 shows that the equation y = 1.1529x + 0.2129 is satisfied, and the correlation coefficient is greater than 0.99.
Table 2.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 2.9 . The Standard Curve of Urate Oxidase
2. The enzyme activity of UAO
Analysis: 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 2.10 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 3. 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 2.10 .The curve uric acid oxidase activity of pGEX-4T-1-UAO
A.Electrophoresis for the target genes (UAA) and the pGEx-4T-1-UAO vectors linearized
Analysis: The length of the target gene a llantoinase is 1485 bp. The length of the target gene allantoicase is 1074 bp. Figure 2.11 A shows that the fragment lengths are consistent with the results. The length of the plasmid pGex-4T-1-UAO is 5301 bp. Figure 13B shows that he fragment lengths are consistent with the results. It indicates that we have successfully amplified the target gene.
Figure 2.11 . Electrophoresis for the genes a llantoinase, allantoicase and pGex-4T-1-UAO
Culture plates for DH5α E.coli transformed with pGex-UAO plasmids
Analysis: we transformed the plasmid pGex-4T-1-UAA into DH5α. Figure 14 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 14C show that the fragment lengths are consistent with the results. In Figure 2.12 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 2.12 . The electrophoresis of the monoclonal antibody in E.coil DH5α
Analysis: 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 2.13 ), without any genetic mutations. It indicates that our plasmid pGex-4T-1-UAA has been successfully constructed.
Figure 2.13 . The sequencing of the allantoicase and allantoinase genes on the pGex-4T-1-UAA plasmid
Protein expression
Analysis: 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 2.14 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 16C and D show that the vector pGex-4T-1-UAA was successfully transformed into E.coil BL21(DE3).
Figure 2.14 . The electrophoresis of the monoclonal antibody for pGex-UAA in BL21(DE3)
Note:
Figure 16A-B: The monoclonal plate pGex-UAA in BL21(DE3)
Figure 16C: The Electrophoresis verification of allantoicase in pGex-UAA (BL21(DE3))
Figure 16D: The Electrophoresis verification of allantoinase in pGex-UAA (BL21(DE3))
Analysis: 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 2.15 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 2.15 . 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 4 and Figure 2.16 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 4. 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 2.16 . The curve of UAA activity
2 .Contrast enzyme activity of UAO and UAA
Analysis: by comparing the levels of uric acid after adding UAA and UAO proteins(Figure 2.17 ), 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 2.17 . The curve of UAA and UAO activity
Other Contributions -- Experiment Troubleshooting Notes
|
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 DH5α 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 DH5α bacteria is inadequate. Which induces results that plasmids are not fully transformed to the DH5α . |
Operate the entire procedure again: select appropriate bacteria solutions of DH5α 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. |
|
The spread of DH5α bacteria’s colonies on solid culture plates is not obvious: There aren’t visible spots that refer to DH5α 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 DH5α 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 fusion protein (urate oxidase, allantoinase and allantoicase). You will further test the protein expression and enzyme activity of pGEX-4T-1-UAO and pGEX-4T-1-UAA in Nissle 1917 to find the optimal expression conditions. You can also validate the ability of pGEX-4T-1-UAO and 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.