Difference between revisions of "Part:BBa K3633003"
 
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[[File:T--Shanghai_SFLS_SPBS--Dopamine Synthesis--Pathway.png|600px|center|thumb|Fig 1. Dopamine Synthesis Pathway]]
 
[[File:T--Shanghai_SFLS_SPBS--Dopamine Synthesis--Pathway.png|600px|center|thumb|Fig 1. Dopamine Synthesis Pathway]]
  
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DDC from pig kidney cell
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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.
  
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Aided in dopamine synthesis
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By merging DDC with J23102 promoter, RBS, HpaB, and HpaC, 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.
  
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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.
 
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By merging DDC with J23102 promoter, RBS and HpaBC, 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==
 
==Experiments & Results==
 
===Successful production in E.coli BL21(DE3) and vibrio natriegens===
 
===Successful production in E.coli BL21(DE3) and vibrio natriegens===
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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.
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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℃.]]
 
[[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℃.]]
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==Sequence & Features==
 
==Sequence & Features==
 
<span class='h3bb'></span>
 
<span class='h3bb'></span>
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==References==
 
==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.
 
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.
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 +
2.Du, X., Li, L., Li, J., Yang, C., Frenkel, N., & Welle, A. et al. (2014). UV-Triggered Dopamine Polymerization: Control of Polymerization, Surface Coating, and Photopatterning. Advanced Materials, 26(47), 8029-8033. doi: 10.1002/adma.201403709
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3.Du, X., Li, L., Behboodi-Sadabad, F., Welle, A., Li, J., & Heissler, S. et al. (2017). Bio-inspired strategy for controlled dopamine polymerization in basic solutions. Polymer Chemistry, 8(14), 2145-2151. doi: 10.1039/c7py00051k
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4.Chen, T., Liu, T., Su, T., & Liang, J. (2017). Self-Polymerization of Dopamine in Acidic Environments without Oxygen. Langmuir, 33(23), 5863-5871. doi: 10.1021/acs.langmuir.7b01127

Latest revision as of 01:57, 27 October 2020


coding sequence of DDC enzyme from pig kidney cell

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.

By merging DDC with J23102 promoter, RBS, HpaB, and HpaC, 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.

2.Du, X., Li, L., Li, J., Yang, C., Frenkel, N., & Welle, A. et al. (2014). UV-Triggered Dopamine Polymerization: Control of Polymerization, Surface Coating, and Photopatterning. Advanced Materials, 26(47), 8029-8033. doi: 10.1002/adma.201403709

3.Du, X., Li, L., Behboodi-Sadabad, F., Welle, A., Li, J., & Heissler, S. et al. (2017). Bio-inspired strategy for controlled dopamine polymerization in basic solutions. Polymer Chemistry, 8(14), 2145-2151. doi: 10.1039/c7py00051k

4.Chen, T., Liu, T., Su, T., & Liang, J. (2017). Self-Polymerization of Dopamine in Acidic Environments without Oxygen. Langmuir, 33(23), 5863-5871. doi: 10.1021/acs.langmuir.7b01127