pAC-94N plasmid大肠杆菌Beta胡萝卜素合成质粒载体BioVector NTCC保藏中心

BioVector^TM pAC-94N plasmid contains crtI, crtY, and crtI genes of Erwinia herbicola (Pantoea agglomerans) Eho10, and produces beta-carotene in E. coli when complemented with a gene encoding geranylgeranyl diphosphate synthase

For better yield of low copy number pAC-based plasmids, grow liquid cultures on a platform shaker at ca. 30 degrees Celsius. When cultures reach early stationary phase, dilute 2-fold with growth medium, add spectinomycin (150 mg/liter), and "amplify" for several hours before harvest. For plasmid selection and maintenance in E. coli, use chloramphenicol at 30 mg/liter.

Vector backbone BioVector^TM pAC-BETA

Backbone size w/o insert (bp)10609

Total vector size (bp)9268

Modifications to backbone Plasmid BioVector^TMpAC-BETA was digested with EcoRV (to delete the N terminal portion of the crtE gene), and a ca. 9.27 kB fragment was then gel-purified and ligated to give pAC-94N.

Vector typelow copy number bacterial cloning vector

Bacterial Resistance(s)Chloramphenicol, 25 μg/mL

Growth Temperature30°C

Growth Strain(s)Top10

Growth instructions Use to confirm the function of the products of genes or cDNAs that are thought to encode a geranylgeranyl diphosphate synthase enzyme, or to screen genomic or cDNA libraries for plasmids containing such genes (crtE) or cDNAs (ggps). The plasmid BioVector^TM pAdGGPS3 can be used as a positive control. Grow in liquid culture on a platform shaker at 28 degrees Celsius in darkness for 2-3 days, or on agar plates at room temperature for 3-7 days. A yellow colony color and an accumulation of beta-carotene is indicative of GGPS activity. Note that a pale yellow color and a trace of beta-carotene will be observed in the absence of a second plasmid with a gene or cDNA encoding a GGPS because the endogenous E. coli farnesyl diphosphate synthase (IspA) produces a small amount of the C20 geranylgeranyl diphosphate in addition to the major C15 product, farnesyl diphosphate.

Copy number Low Copy

Gene/Insert namecrtY, crtI, crtB

SpeciesErwinia herbicola Eho10

GenBank IDM87280.1

Promoterendogenous promoters

Cloning methodRestriction Enzyme

5′ cloning siteEcoRV (not destroyed)

3′ cloning siteEcoRV (not destroyed)

BioVector^TM pAC-94N, Plasmid 53281. Gene/Insert:crtY, crtI, crtB (Other)