pAtipiTrc
(Plasmid #53275)

• Purpose: Contains an Arabidopsis thaliana idi1 cDNA fused to a strong Trc promoter. Produces an active isopentenyl diphosphate isomerase (IDI) in E. coli.
• Depositing Lab: Francis X Cunningham Jr
• Publication: Cunningham FX et al J Bacteriol. 2000 Oct;182(20):5841-8.

Backbone

• Vector backbone: pBluescript SK-
• Backbone manufacturer: Stratagene
• Backbone size w/o insert (bp): 2958
• Total vector size (bp): 4553
• Modifications to backbone: A 0.85 kB KpnI-EcoRI fragment containing most of an Arabidopsis ipi1 cDNA [Cunningham, FX Jr, Gantt E (2000) Identification of Multi-Gene Families Encoding Isopentenyl Diphosphate Isomerase in Plants by Heterologous Complementation in Escherichia coli. Plant Cell Physiol. 41, 119-123.] was excised from the original cDNA library plasmid and ligated in the corresponding sites of plasmid pTrcHis B. Subsequent digestion with NcoI and XhoI, filling-in with the Klenow fragment of DNA polymerase, and ligation to recircularize, yielded a plasmid in which the coding region of ipi1, lacking the first 48 codons, is fused in frame to the strong bacterial Trc promoter and 6 additional codons that specify the amino acids MSRSAA. An EcoRV-EcoRI fragment, containing the cDNA together with the Trc promoter, was excised, gel-purified, and ligated in the corresponding sites of pBluescript SK− to produce the plasmid pAtipiTrc (also referred to as clone 11-2).
• 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: This plasmid produces an active isopentenyl diphosphate isomerase (IPI) enzyme in E. coli, and can be used to enhance carotenoid production in E. coli containing carotenoid-producing plasmids, such as pAC-LYC and pAC-BETA and pAC-ZEAX, that lack a gene encoding an IPI. Use as a positive control when examining the activity of the products of prospective IPI-encoding genes or cDNAs in E. coli strain TOP10 (most other E. coli strains also are suitable) containing pAC-LYC or pAC-BETA. In this case, 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. An active IPI enzyme will result in E. coli colonies much darker in color.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: ipi1
• Alt name: Atdyi
• Alt name: ipiAt1
• Species: A. thaliana (mustard weed)
• Insert Size (bp): 1595
• GenBank ID: AF188066.1
• Entrez Gene: IPP2 (a.k.a. AT3G02780, ATISOPENTENYL DIPHOSPHE ISOMERASE 2, F13E7.28, F13E7_28, IDI2, IPIAT1, ISOPENTENYL PYROPHOSPHATE:DIMETHYLLALLYL PYROPHOSPHATE ISOMERASE, isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase 2)
• Promoter: Trc

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: EcoRV (not destroyed)
• 3′ cloning site: EcoRI (not destroyed)
• 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