pAC-GAMMAipi
(Plasmid #53278)

• Purpose: Has ctrE, crtB, crtI, crtY, and idi genes of Erwinia herbicola (Pantoea agglomerans) Eho10 and crtYm gene of marine bacterium P99-3. and produces gamma-carotene in Escherichia coli
• Depositing Lab: Francis X Cunningham Jr
• Publication: Cunningham FX et al Plant J. 2005 Feb;41(3):478-92.

Backbone

• Vector backbone: pAC-LYCipi
• Backbone manufacturer: Francis X. Cunningham, Jr.
• Backbone size w/o insert (bp): 8913
• Total vector size (bp): 11586
• Modifications to backbone: A 2.75 kb PvuII-AseI fragment containing the crtYm gene of the marine bacterium P99-3 was excised from plasmid pBS603dDOA [Teramoto, M., Takaichi, S., Inomata, Y., Ikenaga, H. and Misawa, N. (2003) Structural and functional analysis of a lycopene beta-monocyclase gene isolated from a unique marine bacterium that produces myxol. FEBS Lett. 545, 120–126.] and inserted in the AseI and Klenow-filled HindIII sites of pAC-LYCipi to yield pAC-GAMMAipi.
• 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 days in darkness for best gamma-carotene production, or grow on agar plates at room temperature for 3-5 days. Longer growth periods result in increased conversion of gamma-carotene (one beta-ring) into beta-carotene (two beta-rings)
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: crtYm
• Alt name: lycopene beta monocyclase
• Species: Marine bacterium P99-3
• Insert Size (bp): 2750
• GenBank ID: AB097813.1
• Promoter: endogenous promoter

Cloning Information

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

Resource Information

• A portion of this plasmid was derived from a plasmid made by: Plasmid pBS603dDOA [Teramoto, M., Takaichi, S., Inomata, Y., Ikenaga, H. and Misawa, N. (2003) Structural and functional analysis of a lycopene beta-monocyclase gene isolated from a unique marine bacterium that produces myxol. FEBS Lett. 545, 120–126.], containing the gene crtYm of the marine bacterium P99-3, was a gift of Norihiko Misawa and Maki Teramoto (both of the Marine Biology Institute, Iwate, Japan).

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.