pAtCHYb1Δ129N
(Plasmid #53367)

• Purpose: Contains a truncated Arabidopsis beta-carotene hydroxylase cDNA, the product of which converts beta-carotene primarily to monocyclic beta-cryptoxanthin in E. coli. Use with pAC-BETA or pAC-BETAipi.
• Depositing Lab: Francis X Cunningham Jr
• Publication: Cunningham FX et al Plant J. 2005 Feb;41(3):478-92.

Backbone

• Vector backbone: pTrcHis A
• Backbone manufacturer: Invitrogen
• Backbone size w/o insert (bp): 4414
• Total vector size (bp): 5062
• Modifications to backbone: A cDNA encoding an Arabidopsis thaliana beta-carotene hydroxylase was excised from the original cDNA library plasmid [see Sun Z, Gantt E, Cunningham FX Jr (1996) Cloning and functional analysis of the beta-carotene hydroxylase of Arabidopsis thaliana. J Biol Chem. 271, 24349-24352.] with PvuII (within the cloning vector upstream of the 5’ end of the cDNA) and NotI (within the adaptor at the 3’ end of the cDNA), blunted using the Klenow enzyme, and ligated in the forward orientation in pTrcHis A that had been digested with BamHI and blunted with the Klenow enzyme. The resulting plasmid, which retained a BamHI site immediately upstream of the cDNA, was digested with BamHI, then partially digested with BglII (for which there was one site within the open reading frame of the cDNA and a second site in the vector immediately after the cDNA), and a polynucleotide of ca. 5 kB was gel purified and self-ligated. The resulting plasmid contains a truncated open reading frame (lacking codons for the first 129 amino acids) for the A. thaliana beta-carotene hydroxylase, fused at the N terminus to two codons that specify the amino acids MG and lying immediately downstream of and partially overlapping (but not fused to) a small open reading frame under the control of the strong Trc promoter. This plasmid is also known as pAtCHYb-BglII [Cunningham FX Jr, Gantt E (2007) A portfolio of plasmids for identification and analysis of carotenoid pathway enzymes: Adonis aestivalis as a case study. Photosynth Res. 92, 245-259.].
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): XL1 Blue
• Growth instructions: Can be used with pAC-BETA or pAC-BETAipi in E. coli strain TOP10 to produce beta-cryptoxanthin. For this purpose, grow liquid cultures on a platform shaker at 28 degrees Celsius for 2-3 days in darkness, or grow on agar plates at room temperature for 3-7 days.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: chyB1
• Alt name: beta-carotene hydroxylase 1
• Species: A. thaliana (mustard weed)
• Insert Size (bp): 622
• Mutation: Deleted codons for the first 129 amino acids
• GenBank ID: U58919.1
• Entrez Gene: BETA-OHASE 1 (a.k.a. AT4G25700, B1, BCH1, BETA CAROTENOID HYDROXYLASE 1, beta-hydroxylase 1, chy1)
• Promoter: Trc

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: BamHI (destroyed during cloning)
• 3′ cloning site: BamHI (destroyed during cloning)
• 5′ sequencing primer: pTrcHis-F
• 3′ sequencing primer: pTrcHis-R

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry