Our Parts
The main goal of our project was to establish the construct which will help us to metabolize 7-ketocholesterol, consisting in the use of three specific enzymes, but for further applications we submitted them in single modules. This will serve as the basis of a future library for standardized work related to atherosclerosis.
Cholesterol Oxidase
This enzyme was first detected in Chromobacterium sp. We introduced it in a plasmid backbone with chloramphenicol resistance: pSB1C3. Its length is of 1871 nucleotides and its codons were optimized in order to use it on E. coli, it already included a stop codon for transcription, it was also modified by the addition of a glycosilation site (NIT) and the peptide signal of human S-cathepsin.
Oxoacyl Reductase
This enzyme was detected in Rhodococcus jostii . We introduced it in a plasmid backbone with chloramphenicol resistance: pSB1C3. Its length is of 1007 nucleotides and its codons were optimized in order to use it on E. coli, it already included a stop codon for transcription, it was also modified by the addition of a glycosilation site (NIT) and the peptide signal of human S-cathepsin.
7-dehydratase
This enzyme (7-alpha dehydratase) was detected in Rhodococcus jostii . We introduced it in a plasmid backbone with chloramphenicol resistance: pSB1C3. Its length is of 602 nucleotides and its codons were optimized in order to use it on E.coli, it already included a stop codon for trancription, it was also modified by the addition of a glycosilation site (NIT) and the peptide signal of human S-cathepsin.
Neomycin Resistance
This selective marker was obtained from an mammalian expression vector. NeoR's length is 855 nucleotides and it was isolated from pcDNA3.1(-)/myc-His A.
BGHPA
Bovine Growth Hormone Polyadenilation Signal for nuclease resistance. Translation terminator for eukaryotic cells.
PCMV
Constitutive promoter from Cytomegalovirus, this promoter works on eukaryotic cells, driving protein expression.