pCambia2300-RUBYGREEN

A lovely new plasmid I designed and constructed by hand, which produces a strong green fluorescent protein when placed in E. coli and Agrobacterium, acting as a tracer for plasmid presence, and carries a RUBY betalain biosynthesis cassette which is transferred into a plant's genome via Agrobacterium tumefaciens to genetically encode the constant and constitutive production of a pretty red pigment similar to the one found in red beets. This plasmid is capable of replicating in E. coli at high copy for easy minipreps and cloning, but also in Agrobacterium as a low copy tDNA vector which allows for the transient and stable transformation of plants with RUBY.

The backbone of this plasmid is the venerable and open source pCambia2300 which confers kanamycin resistance in both bacteria and plants. The MCS of this plasmid is occupied by an open source superfolder green fluorescent protein, FuGFP, expression cassette which can be cloned out via EcoR1 and Kpn1 restriction sites. The RUBY cassette is cloned in such a way as to be outside the MCS allowing for both the presence of RUBY as well as your other gene of interest with the need to serially clone parts.

Please refer to my Plasmid Library for the sequence data of this plasmid.

For a protocol on how to use this plasmid for the transient expression of RUBY in petunia petals, please see my Protocols Library for step-by-step instructions.

For the latest out of my research projects and hints at potential new products, check out my Open Lab Notebook, where all my work lives.

The plasmid is shipped pre-transformed in a safe non-pathogenic laboratory line of E. coli in an agar stab. Please store at 4C in a fridge or at room temp in the dark. DO NOT FREEZE. Properly stored unopened agar stabs have lasted well over 5 years with good recovery in my lab.

To maintain the plasmid in the E. coli it comes in, please use standard LB Agar with 100mg/L Kanamycin and incubate overnight at 37C. Please restreak when you would like to use the cells and only work with freshly grown well isolated colonies. Do not grow cells in liquid media directly as it will carry over stressed cells and lead to uneven colony sizes and GFP tracer brightness.

To visualize the FuGFP tracer protein, please use a 395nm or 365nm (ideal) UV light source. I use cheap UV flashlights on Amazon and recommend this brand as its rechargeable, focusable, and durable.

Note that if you wish to transform plants with this plasmid, you must source the Agrobacterium tumefaciens cells yourself. I do not offer Agrobacterium cells for sale at this time. I recommend the GV3101 strain for any work in dicots and LBA4404 for work in monocots. There are a few suppliers including IntactGenomics that sell competent agrobacterium strains, which are expensive, but you only need to purchase it once as long as you maintain the cell line via glycerol or agar stab stocks.

pCambia2300-RUBYGREEN © 2024 by Sebastian S. Cocioba is licensed under Creative Commons Attribution-ShareAlike 4.0 International

 

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1. Acknowledgment of Risk: You acknowledge that the purchase and use of the plasmids involve known and unanticipated risks, which could result in physical or chemical damage, and you understand such potential risks.

2. Assumption of Risk: You knowingly, voluntarily, and expressly waive any claim you may have against the Seller for any such damages sustained as a result of purchasing or using the plasmids.

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