pAC-ZETAipi
(Plasmid #53284)

• Purpose: Contains crtE, crtB, and idi genes of Erwinia herbicola (Pantoea agglomerans) Eho10, and pds (crtP) gene of Synechococcus PCC7942, and thereby produces zeta-carotene 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-EHER
• Backbone manufacturer: Francis X. Cunningham, Jr.
• Backbone size w/o insert (bp): 13581
• Total vector size (bp): 8352
• Modifications to backbone: Plasmid pAC-EHER was digested with DrdI and partially digested with DraIII, a 9.46 kB fragment was gel-purified, the ends were made blunt with the Klenow enzyme, and the fragment was ligated to give pAC-BETA-crtX. Plasmid pAC-ZETAipi was constructed by excising a ca. 2.25 kB PvuII–BamHI fragment containing the Synechococcus sp PCC7942 phytoene desaturase gene, pds (crtP), fused to the N terminal portion of the lacZ gene with promoter, from pPDSdel35 [Linden H, Misawa N, Chamovitz D, Pecker I, Hirschberg J, Sandmann G (1991) Functional complementation in Escherichia coli of different phytoene desaturase genes and analysis of accumulated carotenes. Z Naturforsch C. 46, 1045-1051.], blunting it with the Klenow enzyme, and ligating it together with a 6.1 kB BglII + Klenow-blunted-SphI fragment obtained from pAC-BETAcrtX (see above).
• 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 zeta-carotene production, or grow on agar plates at room temperature for 3-7 days. The pAC-ZETAipi plasmid produces a mixture of cis isomers of zeta-carotene in E. coli, predominantly 9,15,9′-tri-cis-zeta-carotene and 9,9′-di-cis-zeta-carotene [see Isaacson T, Ohad I, Beyer P, Hirschberg J (2004) Analysis in vitro of the enzyme CRTISO establishes a poly-cis-carotenoid biosynthesis pathway in plants. Plant Physiol. 136, 4246-4255.]
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: pds
• Alt name: crtP
• Species: Synechococcus PCC7942
• Insert Size (bp): 2249
• GenBank ID: X55289.1
• Promoter: lac

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: SphI (destroyed during cloning)
• 3′ cloning site: BglII (destroyed during cloning)
• 5′ sequencing primer: none
• 3′ sequencing primer: none

Resource Information

• A portion of this plasmid was derived from a plasmid made by: Plasmid pPDSdel35 [Linden H, Misawa N, Chamovitz D, Pecker I, Hirschberg J, Sandmann G (1991) Functional complementation in Escherichia coli of different phytoene desaturase genes and analysis of accumulated carotenes. Z Naturforsch C. 46, 1045-1051.] was obtained from Dr. Joseph Hirschberg of the Hebrew University of Jerusalem.
• 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.