Difference between revisions of "Part:BBa K3711043"
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This is a composite component for expressing crtB outside the cell. CrtB is transcribed and translated into phytoene synthase，which is the key enzyme for the synthesis of curcumin. It participates in the transformation from two GGPP molecules to octahydrolycopene. AOX1 promoter is a strong promoter induced by methanol. Under the condition of methanol induction, with the help of α factor, crtB is translated and excreted from the cell. | This is a composite component for expressing crtB outside the cell. CrtB is transcribed and translated into phytoene synthase，which is the key enzyme for the synthesis of curcumin. It participates in the transformation from two GGPP molecules to octahydrolycopene. AOX1 promoter is a strong promoter induced by methanol. Under the condition of methanol induction, with the help of α factor, crtB is translated and excreted from the cell. | ||
<h1>'''Usage and Biology'''</h1> | <h1>'''Usage and Biology'''</h1> | ||
| − | CrtB is derived from Erwinia and encodes octahydrolycopene synthase (PSY), which is involved in the synthesis of carotenoids. The early steps of carotenoid biosynthesis pathway include the synthesis of Geranylgeranyl pyrophosphate (GGPP), the condensation of two molecules of GGPP to octahydrolycopene, and desaturation of octahydrolycopene into plant fluorene, β-carotene, protolycopene and lycopene. crtB encodes octahydrolycopene synthase, which is responsible for the condensation of two molecules of GGPP into octahydrolycopene. | + | CrtB is derived from Erwinia and encodes octahydrolycopene synthase (PSY), which is involved in the synthesis of carotenoids. The early steps of carotenoid biosynthesis pathway include the synthesis of Geranylgeranyl pyrophosphate (GGPP), the condensation of two molecules of GGPP to octahydrolycopene, and desaturation of octahydrolycopene into plant fluorene, β-carotene, protolycopene and lycopene. crtB encodes octahydrolycopene synthase, which is responsible for the condensation of two molecules of GGPP into octahydrolycopene. <br> |
Carotenoids are widely found in nature. More than 630 different natural carotenoids have been identified. They are de novo synthesized from isoprene-like precursors, only in photosynthetic organisms and some microorganisms. The synthesis of carotenoids is encoded by plasmids or chromosome genes. The genes that encode carotenoid biosynthesis are clustered in a 12.4kb fragment. Genetic studies have shown that the expression of these genes requires CAMP. | Carotenoids are widely found in nature. More than 630 different natural carotenoids have been identified. They are de novo synthesized from isoprene-like precursors, only in photosynthetic organisms and some microorganisms. The synthesis of carotenoids is encoded by plasmids or chromosome genes. The genes that encode carotenoid biosynthesis are clustered in a 12.4kb fragment. Genetic studies have shown that the expression of these genes requires CAMP. | ||
<h1>'''Molecular cloning'''</h1> | <h1>'''Molecular cloning'''</h1> | ||
Revision as of 08:50, 21 October 2021
AOX1-α factor-crtB-AOX1 Terminator
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1187
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1919
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1913
Description
This is a composite component for expressing crtB outside the cell. CrtB is transcribed and translated into phytoene synthase，which is the key enzyme for the synthesis of curcumin. It participates in the transformation from two GGPP molecules to octahydrolycopene. AOX1 promoter is a strong promoter induced by methanol. Under the condition of methanol induction, with the help of α factor, crtB is translated and excreted from the cell.
Usage and Biology
CrtB is derived from Erwinia and encodes octahydrolycopene synthase (PSY), which is involved in the synthesis of carotenoids. The early steps of carotenoid biosynthesis pathway include the synthesis of Geranylgeranyl pyrophosphate (GGPP), the condensation of two molecules of GGPP to octahydrolycopene, and desaturation of octahydrolycopene into plant fluorene, β-carotene, protolycopene and lycopene. crtB encodes octahydrolycopene synthase, which is responsible for the condensation of two molecules of GGPP into octahydrolycopene.
Carotenoids are widely found in nature. More than 630 different natural carotenoids have been identified. They are de novo synthesized from isoprene-like precursors, only in photosynthetic organisms and some microorganisms. The synthesis of carotenoids is encoded by plasmids or chromosome genes. The genes that encode carotenoid biosynthesis are clustered in a 12.4kb fragment. Genetic studies have shown that the expression of these genes requires CAMP.
Molecular cloning
Plasmid with target gene is transformed into E.coli. From them, we acquire large amount of target gene using as raw material for further operation.
- tu**
Using E.coli for amplification, we extract and digest them with Bgl I or Sal I to get linear plasmid, which could be integrated into yeast genome to avoid getting lost while being frozen. Then, concentration of linear plasmid is also applied to achieve higher copy number and higher expression level. Several rounds of electroporation later, we successfully get all the plasmid with AOX1 as promoter into yeast.
- tu**