Purpose
In recent years, energy problems are becoming serious and bio batteries are attracting attention as one of the next generation renewable energy sources. An organism has an enzyme that catalyzes the oxidation-reduction reaction of various substances, and energy is obtained by converting redox reaction energy by the catalytic function of this enzyme. Bio batteries are batteries using enzymes that are biocatalysts. On the anode side, electrons obtained from redox enzymes from sugars and alcohols as substrates move to the cathode side through the electrode and the cathode side enzyme receiving the electrons operates by reducing the substrate. But the disadvantage of bio batteries is their low output. I am studying the cathode of a bio battery. It is important to orientate the enzyme in order to increase the output of the bio cell. For that purpose I focused on the thiol group of cysteine. Since the thiol group can specifically bind to the gold electrode, the enzyme can be oriented and immobilized. Therefore, I use a genetic engineering technique to construct an enzyme that introduced cysteine by changing the base sequence of the wild-type enzyme. Specifically, one nucleotide sequence of a wild-type enzyme is substituted to introduce cysteine.
experimental method
The template plasmid was amplified by inverse PCR. PCR products were subjected to restriction enzyme treatment and self-ligation was carried out. After that, E. coli was transformed, and the obtained colonies were picked up. After culturing, plasmid extraction was carried out and the nucleotide sequence was confirmed.
Result
The amino acid sequence of the plasmid into which the mutation was introduced is shown.
wild type …ATFLVEGKGEAVPVEAL…
mutant … ATFLVEGKGCAVPVEAL…
Wild type indicates the wild-type enzyme plasmid.
Mutant shows the plasmid of the enzyme into which cysteine has been introduced.
Successfully constructed a plasmid in which E (glutamic acid) was replaced with C (cysteine).
Since the thiol group of the substituted cysteine is fixed in orientation to the gold electrode, it may lead to the development of a bio-cell with increased output in the future.
In recent years, energy problems are becoming serious and bio batteries are attracting attention as one of the next generation renewable energy sources. An organism has an enzyme that catalyzes the oxidation-reduction reaction of various substances, and energy is obtained by converting redox reaction energy by the catalytic function of this enzyme. Bio batteries are batteries using enzymes that are biocatalysts. On the anode side, electrons obtained from redox enzymes from sugars and alcohols as substrates move to the cathode side through the electrode and the cathode side enzyme receiving the electrons operates by reducing the substrate. But the disadvantage of bio batteries is their low output. I am studying the cathode of a bio battery. It is important to orientate the enzyme in order to increase the output of the bio cell. For that purpose I focused on the thiol group of cysteine. Since the thiol group can specifically bind to the gold electrode, the enzyme can be oriented and immobilized. Therefore, I use a genetic engineering technique to construct an enzyme that introduced cysteine by changing the base sequence of the wild-type enzyme. Specifically, one nucleotide sequence of a wild-type enzyme is substituted to introduce cysteine.
experimental method
The template plasmid was amplified by inverse PCR. PCR products were subjected to restriction enzyme treatment and self-ligation was carried out. After that, E. coli was transformed, and the obtained colonies were picked up. After culturing, plasmid extraction was carried out and the nucleotide sequence was confirmed.
Result
The amino acid sequence of the plasmid into which the mutation was introduced is shown.
wild type …ATFLVEGKGEAVPVEAL…
mutant … ATFLVEGKGCAVPVEAL…
Wild type indicates the wild-type enzyme plasmid.
Mutant shows the plasmid of the enzyme into which cysteine has been introduced.
Successfully constructed a plasmid in which E (glutamic acid) was replaced with C (cysteine).
Since the thiol group of the substituted cysteine is fixed in orientation to the gold electrode, it may lead to the development of a bio-cell with increased output in the future.