What I did was an easy-bake transient expression plasmid to test out my gene gun using CometGFP that I bought from them. I designed some primers to amp out the gfp and added the ncoI cut site which conveniently begets the plant kozak consensus (eukaryotic rbs) and drove the gene with the cauliflower mosaic virus promoter x2. I got decent expression with it without tweaking or codon optimization. If he just needs a source of gfp, and for some reason can't find a lab that's willing to mail him an egfp cassette, this is a viable option. The company will send you a link to their genbank page upon request after purchase.
To be honest, the agrobacterium transformation plasmids contain resistance markers taken directly from bacteria (ecoli transposon for kanamycin nptII) without modification and is expressed and functions as intended in plants. I wouldn't worry too much about codon optimization. With fluorophores, much like peoples attempts at making gfp beer, ph should be taken into consideration. Plants, based on their media requirements alone, prefer a lower ph than ecoli thus folding may be an issue once the protein is expressed. A crude experiment would be to try to grow ecoli expressing gfp on plates ranging in pH and buffered with MES to stabilize around plant media pH and see what happens. Then again, the ph may just hinder ecoli growth and development thus making this experiment crappy. Pardon the mind vomit...
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
To be honest, the agrobacterium transformation plasmids contain resistance markers taken directly from bacteria (ecoli transposon for kanamycin nptII) without modification and is expressed and functions as intended in plants. I wouldn't worry too much about codon optimization. With fluorophores, much like peoples attempts at making gfp beer, ph should be taken into consideration. Plants, based on their media requirements alone, prefer a lower ph than ecoli thus folding may be an issue once the protein is expressed. A crude experiment would be to try to grow ecoli expressing gfp on plates ranging in pH and buffered with MES to stabilize around plant media pH and see what happens. Then again, the ph may just hinder ecoli growth and development thus making this experiment crappy. Pardon the mind vomit...
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
From: Mike Horwath
Sent: 10/15/2014 10:43 AM
To: diybio@googlegroups.com
Cc: scocioba@gmail.com; stygianfyre14@gmail.com; cathalgarvey@cathalgarvey.me
Subject: Re: [DIYbio] Re: Plant transformation using Agrobacteirum
Looks like they are currently selling the four "IP-free" fluorophores as E.coli expression plasmids, although that would not be so useful for plant expression. The sequences can be found at Biobricks.
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