Re: [DIYbio] Prokaryote genes into Eukaryote; Biotech

depends how familiar you already are with bio, sterile technique,
etc... your budget is going to be between $1000-$5000 USD I think

if you already know how to reliably grow your mushroom-of-interest,
that's good because a lot of the experiments will make growth
efficiency suffer, so you need to start with well-growing cultures to
begin.

it would probably be wise to start off by finding if there is any DNA
sequenced in your target organism, and then see if homologous
recombination might work (or if its been done before in that
organism)... if homologous recombination won't work, then you need to
look for a transposon that will work. You could also look for small
plasmids that might work in the organism, you can check the organism
itself by extracting DNA and doing a plasmid prep, then gel
electrophoresing the resulting sample to see if there is a gel band
that is a few 1000 bases.

If there's a plasmid, then great, you can isolate that as I mentioned
above, and either have it sequenced fully or map it with restriction
enzymes (that would be a lot more PITA than just paying to get the
thing sequenced). Then with the sequence you'd need to try and
identify if there are genes and promoters and such on the plasmid...
if there's a promoter, you can try replicating it with PCR, and then
adding your GFP to that, then reinserting the promoter:GFP back into
the plasmid (you would need to add a selective marker, i.e. an
antibiotic resistance gene), and then insert your newly modified
plasmid back into the organism.

If homologous recombination hasn't been done or doesn't look like it
will work, and there's no natural plasmid, and you can't find a
transposon documented... you will have to go a different and possibly
even more complicated route.

The 21st century way to do it though, is to start by getting the whole
genome sequenced. BGI (beijing genome institute) may do it for free,
if they haven't seen the organism before, and if it's of interest to
the scientific community to have the genome of said organism.

On Mon, Aug 27, 2012 at 6:40 AM, Tim Persaud <timmypersaud@hotmail.com> wrote:
> Does anyone know if it's possible for a home scientist to insert prokaryotic
> genes into eukaryotes.
>
> Let's say gfp from a bacteria into mushroom spores?
> Wow, the fact that they're spores first could present a problem....
>
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--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

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