PurposeContains 3 genes (crtE, crtI, and crtB) of the carotenoid pathway gene cluster of Erwinia herbicola (Pantoea agglomerans) Eho10 and thereby produces lycopene in Escherichia coli
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||53270||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) 9476
Modifications to backboneConstructed from pAC-EHER by deletion of adjacent 0.8 and 1.1 kB BamHI-BamHI frag- ments, as well as and adjacent 1.6 and 0.6 kB AvaI-AvaI fragments. This served to remove most or all of the coding regions of the idi (ORF6), crtX, crtY, and crtZ genes.
Vector typelow copy number bacterial cloning vector
Growth in Bacteria
Growth instructionsGrow liquid cultures on a platform shaker at 28 degrees Celsius for 2-3 days in darkness for best lycopene production, or grow on agar plates at room temperature for 3-7 days. Use to confirm the function of the products of prospective DXS or IPI or LytB-encoding genes or cDNAs. Active enzymes will yield E. coli colonies much darker in color [see Cunningham FX Jr, Lafond TP, Gantt E (2000) Evidence of a role for LytB in the nonmevalonate pathway of isoprenoid biosynthesis. J Bacteriol. 182, 5841-5848.]. Use pAtipiTrc or pdxs/ipiTrc as a positive control.
Gene/Insert namecrtE, crtI, crtB
SpeciesErwinia herbicola Eho10
- Promoter endogenous promoters
- Cloning method Restriction Enzyme
- 5′ cloning site NA (unknown if destroyed)
- 3′ cloning site NA (unknown if destroyed)
- 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-LYC was a gift from Francis X Cunningham Jr (Addgene plasmid # 53270 ; http://n2t.net/addgene:53270 ; RRID:Addgene_53270)
For your References section:Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942. Cunningham FX Jr, Sun Z, Chamovitz D, Hirschberg J, Gantt E. Plant Cell. 1994 Aug;6(8):1107-21. 10.1105/tpc.6.8.1107 PubMed 7919981