pAC-94N
(Plasmid #53281)

• Purpose: 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
• Depositing Lab: Francis X Cunningham Jr
• Publication: Cunningham FX et al Photosynth Res. 2007 May;92(2):245-59. Epub 2007 Jul 17.

Backbone

• Vector backbone: pAC-BETA
• Backbone manufacturer: Francis X. Cunningham, Jr.
• Backbone size w/o insert (bp): 10609
• Total vector size (bp): 9268
• Modifications to backbone: Plasmid pAC-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 type: low copy number bacterial cloning vector

Growth in Bacteria

• Bacterial Resistance(s): Chloramphenicol, 25 μg/mL
• Growth Temperature: 30°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 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

• Gene/Insert name: crtY, crtI, crtB
• Species: Erwinia herbicola Eho10
• GenBank ID: M87280.1
• Promoter: endogenous promoters

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: EcoRV (not destroyed)
• 3′ cloning site: EcoRV (not destroyed)
• 5′ sequencing primer: none
• 3′ sequencing primer: none

Resource Information

• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

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.