Difference between revisions of "Part:BBa K3633012"
Line 17: | Line 17: | ||
They added 0.1 mM IPTG for induction. Again, after 20 h culture at 37℃, 220 rpm, we centrifuged the cells, discarded the LB medium, and resuspended the cell pellet in sterilized water. They then cultured the cells at 20℃, 120 rpm for 102 h and acquired expected results. They did not test the production in Vibrio natriegens because the bacterial strain we acquired (ATCC 14048) does not have the T7 promoter. | They added 0.1 mM IPTG for induction. Again, after 20 h culture at 37℃, 220 rpm, we centrifuged the cells, discarded the LB medium, and resuspended the cell pellet in sterilized water. They then cultured the cells at 20℃, 120 rpm for 102 h and acquired expected results. They did not test the production in Vibrio natriegens because the bacterial strain we acquired (ATCC 14048) does not have the T7 promoter. | ||
− | [[File:T--Shanghai_SFLS_SPBS--Betalains Result.png|600px|center|thumb|Fig 2. | + | [[File:T--Shanghai_SFLS_SPBS--Betalains Result.png|600px|center|thumb|Fig 2. Production of dopaxanthin and indoline-betacyanin in E. coli BL21(DE3) after induction. (A) Production of dopaxanthin, after resuspension in sterilized water at 20℃, 120 rpm in 102 h. The horizontal axis is time (hours), and the vertical axis is absorbance of the bacterial solution at 415 nm. (B) Production of indoline-betacyanin, after resuspension in sterilized water at 20℃, 120 rpm in 102 h. The horizontal axis is time (hours), and the vertical axis is absorbance of the bacterial solution at 525 nm. (C) Production of dopaxanthin from 0-102 h. (D) Production of indoline-betacyanin from 0-102 h.]] |
==Sequence & Features== | ==Sequence & Features== |
Revision as of 09:47, 24 October 2020
A composite part to express 4,5-DODA induced by IPTG, responsible for production of betalains
Description
c
Betalains are water-soluble nitrogen-containing pigments that are subdivided in red-violet betacyanins and yellow-orange betaxanthins. Due to glycosylation and acylation betalains exhibit a huge structural diversity. Betanin (betanidin-5-O--glucoside) is the most common betacyanin in the plant kingdom.
The biosynthesis of betalains in plants excludes that of anthocyanins. During the biosynthesis of betalains in the cytoplasm three enzymes are involved: Tyrosinase, 4,5-DOPA-extradioldioxygenase, and betanidin-glucosyltransferase. The amino acid L-tyrosine, which is enzymatically formed over the shikimate pathway from arogenic acid, is the precursor for the biosynthesis of L-DOPA. Tyrosine is hydroxylated by means of the enzyme tyrosinase to DOPA (I) that is formed to betalamic acid or to cyclo-DOPA. The biosynthesis of betalamic acid, which is the basic structure of betalains as follow: 4,5-DOPA-extradiol dioxygenase opens the cyclic ring of L-DOPA between carbons 4 and 5, thus producing 4,5-seco-DOPA (II). This intermediate product occurs naturally. Due to spontaneous intramolecular condensation between the amine group and the aldehyde group of 4,5-seco-DOPA betalamic acid is formed.
In order to make the production of the Betacyanin, Shanghai_SFLS_SPBS built the biobrick with 4,5-DODA and T7 promoter and added the substrate L-Dopa and 0.1mM IPTG to induce the promoter. The 4,5-seco-DOPA will spontaneously convert into Betalamic acid with the help of ascorbic acid(Vitamin C).And Dopaxanthin/Indoline-Betacyanin will be subsequently synthesized by adding the substrate of L-DOPA/Indoline. The biobrick was successfully expressed in E.coli BL21(DE3). The two kind of pigments were produced and application of the hair-dye process was successful as well.
Experiments & Results
Successful production in E.coli BL21(DE3)
The team members constructed the plasmid T7-4,5-DODA using Gibson Assembly. They added 0.1 mM IPTG for induction. Again, after 20 h culture at 37℃, 220 rpm, we centrifuged the cells, discarded the LB medium, and resuspended the cell pellet in sterilized water. They then cultured the cells at 20℃, 120 rpm for 102 h and acquired expected results. They did not test the production in Vibrio natriegens because the bacterial strain we acquired (ATCC 14048) does not have the T7 promoter.
Sequence & Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]