Part:BBa_K3711043
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.
The bands of AOX1-α factor-FMO-AOX1 Terminator (3000+bp), AOX1-α factor-crtE-AOX1 Terminator (almost 3000bp), AOX1-α factor-crtB-AOX1 Terminator (less than 3000bp) and AOX1-α factor-crtI-AOX1 Terminator (3000+bp) from colony PCR are identical to the theoretical lengths of 3214bp, 2746bp, 2767bp and 3316 bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid had successfully transformed into E.coli.
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.
The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast.
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