Part:BBa_K3711071
Panb1-crtB-AOX1 Terminator
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 124
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1108
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1102
Description
This is a composite part for intracellular expression of crtB. Panb1 is a constitutive promoter in yeast, which is expressed under anaerobic conditions, while under aerobic conditions, Panb1, as a repression target of ROX1, is inhibited. When Panb1 initiates the expression, crtB is expressed and participates in the production from two GGPP molecules to octahydrolycopene.
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
Not quite to what we expect, after repeated transfection to the yeast, only a few products are expressed inside of eukaryotic system. Because of the large molecular weight and various types of some of our protein, we suspect that the common signal peptide we use, α-factor, is not enough to bring our protein out of the cell. While there is some of the genes without detectable products and we are hoping to get higher expression level, new primers for PCR are designed to ignore α-factor from our target gene in PCR. Then, likewise, we reconstruct this series of plasmid without α-factor through similar double-enzyme digestion and reconnection which insert our target genes right behind Panb1 promoter.
The bands of Panb1-crtB-AOX1 Terminator (less than 2000bp), Panb1-crtE-AOX1 Terminator (less than 2000bp) and Panb1-FMO-AOX1 Terminator (2500bp) from colony PCR are identical to the theoretical lengths of 1859bp, 1838bp and 2437bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid are successfully constructed.
To solve this, we reconstruct plasmids without the signal peptide and try to do intracellular expression. This is aim at all the undetectable or low-expressed genes.
The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast. Target genes are confirmed exist in the yeast of multiple bands, which could be the result of polluted electroporation cup.
SDS-PAGE
After verification of successful transfection, we can’t test the protein directly due to intracellular expression. So, we extract the total protein in yeast and go for a purification through Nickel-affinity chromatography column, then apply SDS-PAGE to separate target protein from the large amount and various type of total protein to confirm whether our target protein could be expressed and value its expression level quantitatively.
Different from impure or permeate bands, the target protein located around 50kDa, bigger than the theoretical 35.30kDa but still within explainable and acceptable range of glycosylation modification. crtB could be confirmed as successfully expressed.
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