pAC-BETA-At
(Plasmid #53288)

• Purpose: Contains crtE, crtB, and crtI genes of Erwinia herbicola (Pantoea agglomerans) Eho10, and an lcyB cDNA of Arabidopsis thaliana. Use with pY2F to produce alpha-carotene in E. coli.
• Depositing Lab: Francis X Cunningham Jr
• Publication: Cunningham FX et al Eukaryot Cell. 2007 Mar;6(3):533-45. Epub 2006 Nov 3.

Backbone

• Vector backbone: pAC-LYC
• Backbone manufacturer: Francis X. Cunningham, Jr.
• Backbone size w/o insert (bp): 9476
• Total vector size (bp): 11304
• Modifications to backbone: The entire open reading frame of an Arabidopsis lcyB cDNA [GenBank U50739.1] in plasmid pZL1 was excised as an EcoRI-HindIII fragment of ca. 1.8 kB and cloned in the same sites of pBluescript SK- so that the cDNA was fused, in frame, to the N terminal portion of the lacZ gene and under the control of the lac promoter. From this plasmid (pAtLCYbSK) a ca. 1.8-kB NotI-NotI fragment containing the lcyB cDNA (without the lac promoter) was excised, made blunt with the Klenow enzyme, and cloned in the Klenow-blunted HindIII site of pAC-LYC. The lcyB cDNA in this 11.3 kB plasmid was ascertained, by restriction enzyme digestion, to be in the forward orientation. Despite the lack of a bacterial promoter, production of the LCYb enzyme is sufficient so that beta-carotene is the major carotenoid in E. coli, but also low enough so that the introduction of a second plasmid with a strongly-expressed lycopene epsilon cyclase cDNA (i.e plasmid pY2F) will yield predominantly alpha-carotene in E. coli.
• 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 beta-carotene production, or grow on agar plates at room temperature for 3-7 days. Combine with plasmid pY2F and grow liquid cultures on a platform shaker at 28 degrees Celsius for 2-3 days in darkness for best alpha-carotene production, or grow on agar plates at room temperature for 3-7 days.
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: lcyB
• Alt name: lycopene beta-cyclase
• Species: A. thaliana (mustard weed)
• Insert Size (bp): 1824
• GenBank ID: U50739.1
• Entrez Gene: LYC (a.k.a. AT3G10230, LYCOPENE BETA-CYCLASE, lycopene cyclase)
• Promoter: none

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.