pAC-aZEA
(Plasmid #53286)

• Purpose: Contains crtE, and crtB genes of Erwinia herbicola (Pantoea agglomerans) Eho10, crtI gene of Rhodobacter capsulatus, and lcyE cDNA of Arabidopsis thaliana and produces alpha-zeacarotene in E. coli
• 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-NEUR
• Backbone manufacturer: Francis X. Cunningham, Jr.
• Backbone size w/o insert (bp): 11849
• Total vector size (bp): 14019
• Modifications to backbone: An Arabidopsis thaliana cDNA encoding the lycopene epsilon-cyclase enzyme was excised from plasmid pY2F, together with most of the lac promoter, as an AseI-PvuII fragment of ca. 2.3 kB, and ligated in pAC-NEUR that had been digested with HindIII, blunted with the Klenow enzyme and then digested with AseI.
• 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 in darkness for 2-3 days for best alpha-zeacarotene 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
• Species: A. thaliana (mustard weed)
• Insert Size (bp): 2284
• GenBank ID: U50738.1
• Entrez Gene: LUT2 (a.k.a. AT5G57030, LUTEIN DEFICIENT 2, LYCOPENE EPSILON-CYCLASE)
• Promoter: lac

Cloning Information

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

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.