Re: [DIYbio] Bioluminescent yogurt (Again!)

On Wed, Apr 4, 2012 at 4:24 AM, medminus9 <harshsethia1989@gmail.com> wrote:
> I am not sure, but doesn't the upp-counterselectable gene replacement system
> requires a plasmid? Or can we just insert a linear construct?

Can you rephrase the first sentence? Part of the construct flanked by
homologous regions can have a selection gene, certainly.

If you're talking about transformant selection, Cathal has had some good ideas.
"
By the way, I have in mind a way to do it that would result in an
antibiotic-resistance-free strain at the end: start by replacing the lac
operon using an antibiotic-selection cassette containing your operon,
selecting on glucose medium instead of lactose/milk medium.

Then perform another transformation to replace the antibiotic cassette,
plating on lactose/milk medium to select for transformants. Result;
chromosomal integration of your fluorescence/bioluminescence operon, and
removal of the antibiotic resistance gene. Because your DNA is on the
chromosome rather than a plasmid, it's unlikely to be lost provided
you've ensured there's little if any DNA repetition, and the
evolutionary cost of keeping it isn't punitive.
"

From (https://groups.google.com/d/msg/diybio/HFstp-wXJqE/ozUQqAxkokIJ)

--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

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