Difference between revisions of "Part:BBa K3739015"

Revision as of 19:08, 21 October 2021

PPO-his

The enzyme efficiently catalyzes the reaction of oxidizing dopaming to dopa quinone. His-tag was added to purify the protein.

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.

Fig.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.

Fig.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 Fig.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 Fig.4.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 10: / Line 10: @@
 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
+'''Fig.1.''' the mechanism diagram of PPO
 ===Usage===
@@ Line 16: / Line 16: @@
 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
+'''Fig.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.
+After we received the synthesized DNA sequence, PCR was conducted to verity the correctness of the plasmid, and the experimental results are shown in Fig.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.
+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 Fig.4.