pAC-CANTHipi
(Plasmid #53301)

• Purpose: Contains crtE, idi, crtI, crtY, and crtB genes of Erwinia herbicola (Pantoea agglomerans) Eho10, and a crtO cDNA of Haematococcus pluvialis fused to a Trc promoter. Produces canthaxanthin 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-BETAipi
• Backbone manufacturer: Francis X. Cunningham, Jr.
• Backbone size w/o insert (bp): 9259
• Total vector size (bp): 11200
• Modifications to backbone: An Haematococcus pluvialis beta-carotene C4 oxygenase cDNA (crtO) was excised from plasmid pHPK [Lotan T, Hirschberg J (1995) Cloning and expression in Escherichia coli of the gene encoding beta-C-4-oxygenase, that converts beta-carotene to the ketocarotenoid canthaxanthin in Haematococcus pluvialis. FEBS Lett. 364, 125-128.] as a SacI–KpnI fragment of ca. 1.7 kB. This fragment was ligated in these same sites in pTrcHis A to give pHpKetoTrcA (unpublished), wherein the crtO cDNA was fused, in frame, to a 6His tag and a Trc promoter. An EcoRV–PstI fragment of 1.94 kB, containing the crtO gene fusion and Trc promoter, was excised from pHpKetoTrc, the ends made blunt with the Klenow enzyme, and this fragment was inserted into HindIII-digested, Klenow-blunted pAC-BETAipi. The insert was in the forward orientation, as ascertained by restriction enzyme digests.
• 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 canthaxanthin production, or grow on agar plates at room temperature for 3-7 days.
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: crtO
• Alt name: bkt
• Alt name: beta-carotene C4 oxygenase
• Alt name: beta-carotene 4-ketolase
• Species: Haematococcus pluvialis
• Insert Size (bp): 1937
• GenBank ID: X86782.1
• Promoter: Trc
• Tag / Fusion Protein:
  • 6His (N terminal on insert)

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

• A portion of this plasmid was derived from a plasmid made by: The plasmid pHPK, containing the crtO gene of Haematococcus pluvialis, [Lotan T, Hirschberg J (1995) Cloning and expression in Escherichia coli of the gene encoding beta-C-4-oxygenase, that converts beta-carotene to the ketocarotenoid canthaxanthin in Haematococcus pluvialis. FEBS Lett. 364, 125-128.] was a gift of Dr. Yossi Hirschberg of the Hebrew University of Jerusalem.
• Articles Citing this Plasmid:
  • 7 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.