Part:BBa_K3711044
AOX1-α factor-crtI-AOX1 Terminator
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1255
Illegal NheI site found at 1684 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2059
Illegal XhoI site found at 1187 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2445
Illegal BsaI.rc site found at 2397
Description
This is a composite component for expressing crtI outside the cell. CrtI is transcribed and translated into phytoene desaturase，which is the key enzyme for the synthesis of curcumin. It participates in the transformation from all-trans plant alkene to all-trans lycopene. AOX1 promoter is a strong promoter induced by methanol. Under the condition of methanol induction, with the help of α factor, crtI is translated and excreted from the cell.
Usage and Biology
crtI, which is derived from Erwinia, encodes octahydrolycopene dehydrogenase (PDS) and participates in the synthesis of carotenoids. The early steps of carotenoid biosynthesis pathway include the synthesis of Geranylgeranyl pyrophosphate (GGPP), the condensation of two GGPP molecules to octahydrolycopene, and then desaturating octahydrolycopene into plant fluorene, β-carotene, protolycopene and lycopene. crtI encodes octahydrolycopene dehydrogenase, which is responsible for the desaturating reaction from trans boyanic alkene to trans lycopene.
The most important function of carotenoid pigments, especially carotene in advanced plants, is the protection from photooxidation. In the pathway of carotenoid synthesis, only crtI can convert octahydrolycopene to lycopene, and catalyze the desaturation from trans botanic alkene to trans lycopene. In this transformation process, lycopene was synthesized through four consecutive desaturations of intermediates of fluorene phytic acid, β-carotene and prolycopene. However, limited information can be obtained on the enzymes and genes of carotenoid biosynthesis, because the enzyme produced by crtI is immediately inactivated by separation from the membrane environment, thus preventing its purification and the subsequent cloning of the gene that encodes it.
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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