Difference between revisions of "Part:BBa K3739015"
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===Usage=== | ===Usage=== | ||
According to the biological characteristics of VnDX, we optimized the codon and added a 6His-tag to construct a sequence suitable for expression in VnDX. We hope that it can successfully express polyphenol oxidase in VnDX and reduce the viscosity characteristics of mussel mucin. | According to the biological characteristics of VnDX, we optimized the codon and added a 6His-tag to construct a sequence suitable for expression in VnDX. We hope that it can successfully express polyphenol oxidase in VnDX and reduce the viscosity characteristics of mussel mucin. | ||
| − | The coding sequence of target gene was inserted into the expression vector | + | The coding sequence of target gene was inserted into the expression vector, and the constructed plasmid was transformed into VnDX to verify its heterologous expression. |
Figure 2 Gene circuit diagram of PPO | Figure 2 Gene circuit diagram of PPO | ||
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====1. Identification==== | ====1. Identification==== | ||
After we received the synthesized DNA sequence, PCR was conducted to verity the correctness of the plasmid, and the experimental results are shown in Figure 3. | After we received the synthesized DNA sequence, PCR was conducted to verity the correctness of the plasmid, and the experimental results are shown in Figure 3. | ||
| + | |||
====2. Purification and Proof of the expression==== | ====2. Purification and Proof of the expression==== | ||
T7 promoter was used in the composite part BBa_ to express PPO-his in VnDX. Then we used GE-AKTA Prime Plus FPLC System to get purified GRHPR protein. We found an apparent protein peak in AKTA FPLC System and correct purified protein. Then, our target bands are obserbed through SDS-PAGE and the result is shown in figure 4. | T7 promoter was used in the composite part BBa_ to express PPO-his in VnDX. Then we used GE-AKTA Prime Plus FPLC System to get purified GRHPR protein. We found an apparent protein peak in AKTA FPLC System and correct purified protein. Then, our target bands are obserbed through SDS-PAGE and the result is shown in figure 4. | ||
Revision as of 16:57, 20 October 2021
PPO-his
Further cloning of the target plasmid can be obtained.
Biology
The polyphenol oxidase we used is a kind of polyphenol oxidase from the Marine mussel itself, which regulates the mucin production of mussel. It can oxidase the dopamine on the side chain of mussel mucin to dopamine quinone, and its catalytic effect can be characterized by the change of viscosity characteristics of mussel mucin.
Figure 1 the mechanism diagram of PPO
Usage
According to the biological characteristics of VnDX, we optimized the codon and added a 6His-tag to construct a sequence suitable for expression in VnDX. We hope that it can successfully express polyphenol oxidase in VnDX and reduce the viscosity characteristics of mussel mucin. The coding sequence of target gene was inserted into the expression vector, and the constructed plasmid was transformed into VnDX to verify its heterologous expression.
Figure 2 Gene circuit diagram of PPO
Characterization
1. Identification
After we received the synthesized DNA sequence, PCR was conducted to verity the correctness of the plasmid, and the experimental results are shown in Figure 3.
2. Purification and Proof of the expression
T7 promoter was used in the composite part BBa_ to express PPO-his in VnDX. Then we used GE-AKTA Prime Plus FPLC System to get purified GRHPR protein. We found an apparent protein peak in AKTA FPLC System and correct purified protein. Then, our target bands are obserbed through SDS-PAGE and the result is shown in figure 4.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]