pAC-DELTA
(Plasmid #53273)

• Purpose: Contains crtE, crtB, and crtI genes of Erwinia herbicola (Pantoea agglomerans) Eho10, together with the lcyE gene of Arabidopsis thaliana and thereby produces delta-carotene in E. coli
• Depositing Lab: Francis X Cunningham Jr
• Publication: Sun et al J Biol Chem. 1996 Oct 4;271(40):24349-52.

Backbone

• Vector backbone: pAC-LYC
• Backbone manufacturer: Francis X. Cunningham, Jr.
• Backbone size w/o insert (bp): 9476
• Total vector size (bp): 12248
• Modifications to backbone: A 1.9-kb SmaI-EcoRV fragment containing an A. thaliana lycopene ϵ cyclase cDNA was excised from plasmid y2 and cloned in the Klenow-blunted BamHI site of pTrcHis A to give pTrc-Atϵ. A 2.5-kb EcoRV-KpnI fragment containing the Trc promoter and the A. thaliana lycopene ϵ cyclase was then excised from pTrc-Atϵ and cloned in the Klenow-blunted HindIII site of pAC-LYC to produce pAC-DELTA. The reasoning behind this cloning strategy is now obscure, but the resulting plasmid is useful, yielding delta-carotene as the predominant carotenoid in E. coli. The orientation of the lycopene epsilon-cyclase cDNA relative to the Trc promoter, or to the p15A origin of replication for this plasmid, are both uncertain, therefore the sequence submitted for this plasmid may not be correct.
• 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: Grow liquid cultures on a platform shaker at 28 degrees Celsius for 2-3 days in darkness for best delta-carotene production, or grow on agar plates at room temperature for 3-7 days.
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: lcyE
• Alt name: lycopene epsilon cyclase
• Alt name: lycopene epsilon-monocyclase
• Species: A. thaliana (mustard weed)
• GenBank ID: U50738.1
• Entrez Gene: LUT2 (a.k.a. AT5G57030, LUTEIN DEFICIENT 2, LYCOPENE EPSILON-CYCLASE)
• Promoter: Trc

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: HindIII (destroyed during cloning)
• 3′ cloning site: HindIII (destroyed during cloning)
• 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.