Part:BBa_K3711072
Panb1-crtI-AOX1 Terminator
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 444
Illegal NheI site found at 873 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1248
Illegal XhoI site found at 124 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1634
Illegal BsaI.rc site found at 1586
Description
This is a composite part for intracellular expression of crtI. 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, crtI is expressed and participates in the production from all-trans plant alkene to all-trans lycopene.
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
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-CUS-AOX1 Terminator (2000+bp), Panb1-ACC-AOX1 Terminator (3000bp), Panb1-4CL-AOX1 Terminator (2500+bp) and Panb1-crtI-AOX1 Terminator (2500bp) from colony PCR are identical to the theoretical lengths of 2158bp, 2832bp, 2688bp 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 60kDa, bigger than the theoretical 55.86kDa but still within explainable and acceptable range of glycosylation modification. crtI could be confirmed as successfully expressed.
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