pAC-BETA-At plasmid大肠杆菌产alpha胡萝卜素质粒载体BioVector NTCC保藏中心

BioVector^TM pAC-BETA-At plasmid Contains crtE, crtB, and crtI genes of Erwinia herbicola (Pantoea agglomerans) Eho10, and an lcyB cDNA of Arabidopsis thaliana. Use with BioVector^TM pY2F to produce alpha-carotene in E. coli.

Low copy number bacterial cloning vector

• Backbone size w/o insert (bp)9476

• Total vector size (bp)11304

• Modifications to backbone
  The entire open reading frame of an Arabidopsis lcyB cDNA [GenBank U50739.1] in plasmid pZL1 was excised as an EcoRI-HindIII fragment of ca. 1.8 kB and cloned in the same sites of pBluescript SK- so that the cDNA was fused, in frame, to the N terminal portion of the lacZ gene and under the control of the lac promoter. From this plasmid (BioVector^TM pAtLCYbSK) a ca. 1.8-kB NotI-NotI fragment containing the lcyB cDNA (without the lac promoter) was excised, made blunt with the Klenow enzyme, and cloned in the Klenow-blunted HindIII site of pAC-LYC. The lcyB cDNA in this 11.3 kB plasmid was ascertained, by restriction enzyme digestion, to be in the forward orientation. Despite the lack of a bacterial promoter, production of the LCYb enzyme is sufficient so that beta-carotene is the major carotenoid in E. coli, but also low enough so that the introduction of a second plasmid with a strongly-expressed lycopene epsilon cyclase cDNA (i.e plasmid pY2F) will yield predominantly alpha-carotene in E. coli.
• Vector type
  low copy number bacterial cloning vector

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• 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 2-3 days in darkness for best beta-carotene production, or grow on agar plates at room temperature for 3-7 days. Combine with plasmid BioVector^TM pY2F and grow liquid cultures on a platform shaker at 28 degrees Celsius for 2-3 days in darkness for best alpha-carotene production, or grow on agar plates at room temperature for 3-7 days.
• Copy number
  Low Copy

BioVector^TM pAC-BETA-At, Plasmid BioVector53288. Gene/Insert:lcyB (Arabidopsis thaliana)