Difference between revisions of "Part:BBa K3633009"
(→Experiments & Results) |
|||
Line 15: | Line 15: | ||
===Successful production in E.coli BL21(DE3) and vibrio natriegens=== | ===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 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. | ||
+ | |||
+ | [[File:T--Shanghai_SFLS_SPBS--Dopamine Result 1.png|600px|center|thumb|Fig 2. Dopamine Synthesis Pathway]] | ||
==Sequence & Features== | ==Sequence & Features== |
Revision as of 08:48, 24 October 2020
A composite part to express HpaBC and DDC, responsible for dopamine production
Description
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 constructing this biobrick including promoter J23102, ribosome binding site, HpaBC and DDC, it 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.
Sequence & Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]