pAC-85b
(Plasmid #53282)

• Purpose: Contains crtE, crtI, crtY genes of Erwinia herbicola (Pantoea agglomerans) Eho10, and will produce beta-carotene in E. coli when complemented with a gene encoding phytoene 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: pACYC184
• Backbone manufacturer: New England BioLabs
• Backbone size w/o insert (bp): 4245
• Total vector size (bp): 9424
• Modifications to backbone: Plasmid pAC-BETA was digested with EcoRI and partially digested with BamHI and a 7.1 kB fragment was gel-purified. Plasmid pACYC184 was digested with EcoRI and BamHI and a 2.4 kB fragment was gel-purified. The two fragments were ligated together to give plasmid pAC-85b.
• 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 phytoene synthase enzyme, or to screen genomic or cDNA libraries for plasmids containing such genes (crtB) or cDNAs (psy). The plasmid pAdPSY1 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 phytoene synthase activity.
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: crtE, crtY, crtI
• Species: Erwinia herbicola Eho10
• Insert Size (bp): 7048
• GenBank ID: M87280.1
• Promoter: endogenous promoters

Cloning Information

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

Resource Information

• Articles Citing this Plasmid:
  • 3 References

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.