Difference between revisions of "Part:BBa K3633002"

Revision as of 01:54, 27 October 2020

Coding sequence for HpaC enzyme in E. coli

Description

Fig 1. Dopamine Synthesis Pathway

Dopamine is a hormone and neurotransmitter that plays a variety of important roles in the brain and body. It is an organic chemical substance in the catecholamine and phenethylamine family synthesized in plants and most animals. The synthesis of dopamine is accomplished by removing the carboxyl group from its chemical precursor L-DOPA molecule. L-DOPA, also known as levodopa and l-3,4-dihydroxyphenylalanine, is an amino acid produced and used in part of the normal biological process of humans and some animals and plants. Humans and some other animals utilize HpaB and HpaC enzymes to synthesize L-DOPA from tyrosine, one of the 20 standard amino acids in humans.

By merging HpaC with J23102 promoter, RBS, HpaB, and DDC, the biobrick is successfully expressed in BL21(DE3) and Vibrio natriegens in the experiment of iGEM20_Shanghai_SFLS_SPBS, and dopamine is successfully produced in E. coli in the presence of L-tyrosine.

Experiments & Results

Successful production in E.coli BL21(DE3) and vibrio natriegens

We successfully constructed the HpaBC-DDC plasmid and transformed it into E. coli BL21(DE3) and Vibrio natriegens. Expression of the enzymes HpaB, HpaC, and DDC was confirmed by SDS-PAGE protein electrophoresis. However, the dopamine produced was often oxidized to polydopamine. We tried to produce dopamine in different conditions, including at different temperatures and different oxygen levels, but all of them failed. We learned that dopamine could polymerize in acidic, basic, as well as high-oxygen conditions.

Fig 2. Production of dopamine in E. coli BL21(DE3) and Vibrio natriegens at 25℃ and 37℃ in 72 h. Top left: E. coli BL21(DE3), 25℃. Top right: E. coli BL21(DE3), 37℃. Bottom left: Vibrio natriegens, 25℃. Bottom right: Vibrio natriegens, 37℃.

Sequence & 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]

References

1. A. Das, A. Verma and K. Mukherjee, "Synthesis of dopamine in E. coli using plasmid-based expression system and its marked effect on host growth profiles", Preparative Biochemistry and Biotechnology, vol. 47, no. 8, pp. 754-760, 2017. Available: 10.1080/10826068.2017.1320291.

@@ Line 7: / Line 7: @@
 [[File:T--Shanghai_SFLS_SPBS--Dopamine Synthesis--Pathway.png|600px|center|thumb|Fig 1. Dopamine Synthesis Pathway]]
-HpaC from E.coli
+Dopamine is a hormone and neurotransmitter that plays a variety of important roles in the brain and body. It is an organic chemical substance in the catecholamine and phenethylamine family synthesized in plants and most animals. The synthesis of dopamine is accomplished by removing the carboxyl group from its chemical precursor L-DOPA molecule. L-DOPA, also known as levodopa and l-3,4-dihydroxyphenylalanine, is an amino acid produced and used in part of the normal biological process of humans and some animals and plants. Humans and some other animals utilize HpaB and HpaC enzymes to synthesize L-DOPA from tyrosine, one of the 20 standard amino acids in humans.
-Aided in dopamine synthesis
+By merging HpaC with J23102 promoter, RBS, HpaB, and DDC, the biobrick is successfully expressed in BL21(DE3) and Vibrio natriegens in the experiment of iGEM20_Shanghai_SFLS_SPBS, and dopamine is successfully produced in E. coli in the presence of L-tyrosine.
-Dopamine is a hormone and neurotransmitter that plays a variety of important roles in the brain and body. It is an organic chemical substance in the catecholamine and phenethylamine family that is synthesized in plants and most animals. The synthesis of dopamine is accomplished by removing the carboxyl group from its chemical precursor L-DOPA molecule. L-DOPA, also known as levodopa and l-3,4-dihydroxyphenylalanine, is an amino acid that is produced and used in part of the normal biological process of humans, as well as some animals and plants. Humans, as well as a portion of the other animals that utilize L-DOPA in their biology, make it via biosynthesis from L-tyrosine, one of the 20 standard amino acids that are used by cells to synthesize proteins.
-By merging HpaC with J23102 promoter, RBS, HpaB and DDC, the biobrick is successfully expressed in BL21(DE3) and vibrio natriegens in the experiment of iGEM20_Shanghai_SFLS_SPBS, and dopamine is successfully produced in the presence of L-tyrosine.
 ==Experiments & Results==
 ===Successful production in E.coli BL21(DE3) and vibrio natriegens===
-We successfully constructed the HpaBC-DDC plasmid and transformed it into E. coli BL21(DE3) and Vibrio natriegens. Expression of the enzymes HpaB, HpaC, and DDC were confirmed by an SDS-PAGE protein electrophoresis. However, the dopamine produced was often oxidized to polydopamine. We tried to produce dopamine in different conditions, including at different temperatures and different oxygen levels, but all of them failed. We learned that dopamine can polymerize in acidic, basic, as well as high-oxygen conditions.
+We successfully constructed the HpaBC-DDC plasmid and transformed it into E. coli BL21(DE3) and Vibrio natriegens. Expression of the enzymes HpaB, HpaC, and DDC was confirmed by SDS-PAGE protein electrophoresis. However, the dopamine produced was often oxidized to polydopamine. We tried to produce dopamine in different conditions, including at different temperatures and different oxygen levels, but all of them failed. We learned that dopamine could polymerize in acidic, basic, as well as high-oxygen conditions.
 [[File:T--Shanghai_SFLS_SPBS--Dopamine Result 1.png|600px|center|thumb|Fig 2. Production of dopamine in E. coli BL21(DE3) and Vibrio natriegens at 25℃ and 37℃ in 72 h. Top left: E. coli BL21(DE3), 25℃. Top right: E. coli BL21(DE3), 37℃. Bottom left: Vibrio natriegens, 25℃. Bottom right: Vibrio natriegens, 37℃.]]