PurposeContains crtE, crtB, and idi genes of Erwinia herbicola (Pantoea agglomerans) Eho10, and thereby produces phytoene in E. coli
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||53283||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
Backbone manufacturerFrancis X. Cunningham, Jr.
- Backbone size w/o insert (bp) 13581
- Total vector size (bp) 6103
Modifications to backbonePlasmid pAC-EHER was digested with DrdI and then partially digested with DraIII, and a 9.46 kB fragment was gel-purified, blunted with the Klenow enzyme, and ligated to give pAC-BETA-CrtX (unpublished). pAC-BETA-CrtX was digested with SphI and BglII, the ends were blunted with mung bean nuclease, and a 6.1 kB fragment was gel-purified and ligated to give pAC-PHYTipi.
Vector typelow copy number bacterial cloning vector
Growth in Bacteria
Growth instructionsGrow liquid cultures on a platform shaker at 28 degrees Celsius in darkness for 2-3 days for best phytoene production, or grow on agar plates at room temperature for 3-7 days.
Copy numberLow Copy
Gene/Insert namecrtE, idi, crtY, crtI, crtB
Alt nameidi also known as ORF6
SpeciesErwinia herbicola Eho10
- Promoter endogenous promoters
- Cloning method Restriction Enzyme
- 5′ cloning site SphI (destroyed during cloning)
- 3′ cloning site BglII (destroyed during cloning)
- 5′ sequencing primer none
- 3′ sequencing primer none (Common Sequencing Primers)
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.
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:pAC-PHYTipi was a gift from Francis X Cunningham Jr (Addgene plasmid # 53283 ; http://n2t.net/addgene:53283 ; RRID:Addgene_53283)
For your References section:A portfolio of plasmids for identification and analysis of carotenoid pathway enzymes: Adonis aestivalis as a case study. Cunningham FX Jr, Gantt E. Photosynth Res. 2007 May;92(2):245-59. Epub 2007 Jul 17. 10.1007/s11120-007-9210-0 PubMed 17634749