title: [Construction and preliminary applications of a Saccharomyces cerevisiae detection plasmid using for screening promoter elements].
abstract: Synthetic biology of natural products is the design and construction of new biological systems by transferring a metabolic pathway of interest products into a chassis. Large-scale production of natural products is achieved by coordinate expression of multiple genes involved in genetic pathway of desired products. Promoters are cis-elements and play important roles in the balance of the metabolic pathways controlled by multiple genes by regulating gene expression. A detection plasmid of Saccharomyces cerevisiae was constructed based on DsRed-Monomer gene encoding for a red fluorescent protein. This plasmid was used for screening the efficient promoters applying for multiple gene-controlled pathways. First of all, eight pairs of primers specific to DsRed-Monomer gene were synthesized. The rapid cloning of DsRed-Monomer gene was performed based on step-by-step extension of a short region of the gene through a series of PCR reactions. All cloned sequences were confirmed by DNA sequencing. A vector named pEASYDs-M containing full-length DsRed-Monomer gene was constructed and was used as the template for the construction of S. cerevisiae expression vector named for pYeDP60-Ds-M. pYeDP60-Ds-M was then transformed into S. cerevisiae for heterologous expression of DsRed-Monomer gene. SDS-PAGE, Western blot and fluorescence microscopy results showed that the recombinant DsRed-Monomer protein was expressed successfully in S. cerevisiae. The well-characterized DsRed-Monomer gene was then cloned into a yeast expression vector pGBT9 to obtain a promoter detection plasmid pGBT9Red. For determination efficacy of pGBT9Red, six promoters (including four inducible promoters and two constitutive promoters) were cloned by PCR from the S. cerevisiae genome, and cloned into pGBT9Red by placing upstream of DsRed-Monomer gene, separately. The fluorescence microscopy results indicated that the six promoters (GAL1, GAL2, GAL7, GAL10, TEF2 and PGK1) can regulate the expression of DsRed-Monomer gene. The successful construction of pGBT9Red lays the foundation for further analysis of promoter activity and screening of promoter element libraries.
authors.affiliation: State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Key Laboratory of Biosynthesis of Natural Products, Ministry of Health of PRC, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.
authors.forename: Zhi-Fang
authors.initials: ZF
authors.lastname: Wang
authors.forename: Zhi-Biao
authors.initials: ZB
authors.lastname: Wang
authors.forename: Li-Na
authors.initials: LN
authors.lastname: Li
authors.forename: A N
authors.initials: AN
authors.lastname: Jian-Mei
authors.lastname: Wang-Wei
authors.forename: Ke-Di
authors.initials: KD
authors.lastname: Cheng
authors.forename: Jian-Qiang
authors.initials: JQ
authors.lastname: Kong
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers / Q000096:biosynthesis; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D058615:Synthetic Biology; D014170:Transformation, Genetic; D000097573:Red Fluorescent Protein
chemical_list: D017931:DNA Primers; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein; C413663:fluorescent protein 583