PurposeContains crtI, crtY, and crtI genes of Erwinia herbicola (Pantoea agglomerans) Eho10, and produces beta-carotene in E. coli when complemented with a gene encoding geranylgeranyl diphosphate synthase
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||53281||Standard format: Plasmid sent in bacteria as agar stab||1||$85|
This material is available to academics and nonprofits only.
Backbone manufacturerFrancis X. Cunningham, Jr.
- Backbone size w/o insert (bp) 10609
- Total vector size (bp) 9268
Modifications to backbonePlasmid pAC-BETA was digested with EcoRV (to delete the N terminal portion of the crtE gene), and a ca. 9.27 kB fragment was then gel-purified and ligated to give pAC-94N.
Vector typelow copy number bacterial cloning vector
Growth in Bacteria
Bacterial Resistance(s)Chloramphenicol, 25 μg/mL
Growth instructionsUse to confirm the function of the products of genes or cDNAs that are thought to encode a geranylgeranyl diphosphate synthase enzyme, or to screen genomic or cDNA libraries for plasmids containing such genes (crtE) or cDNAs (ggps). The plasmid pAdGGPS3 can be used as a positive control. Grow in liquid culture on a platform shaker at 28 degrees Celsius in darkness for 2-3 days, or on agar plates at room temperature for 3-7 days. A yellow colony color and an accumulation of beta-carotene is indicative of GGPS activity. Note that a pale yellow color and a trace of beta-carotene will be observed in the absence of a second plasmid with a gene or cDNA encoding a GGPS because the endogenous E. coli farnesyl diphosphate synthase (IspA) produces a small amount of the C20 geranylgeranyl diphosphate in addition to the major C15 product, farnesyl diphosphate.
Copy numberLow Copy
Gene/Insert namecrtY, crtI, crtB
SpeciesErwinia herbicola Eho10
- Promoter endogenous promoters
- Cloning method Restriction Enzyme
- 5′ cloning site EcoRV (not destroyed)
- 3′ cloning site EcoRV (not destroyed)
- 5′ sequencing primer none
- 3′ sequencing primer none (Common Sequencing Primers)
Terms and Licenses
- Not Available to Industry
- Zeocin® is an InvivoGen trademark.
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-94N was a gift from Francis X Cunningham Jr (Addgene plasmid # 53281 ; http://n2t.net/addgene:53281 ; RRID:Addgene_53281)
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