Thanks for the heads up, Darrell. Would the methylase have to be transformed in a separate plasmid so that it has time to act before introduction of the restriction enzyme, or are they fast-acting enough to be transformed on one plasmid? I suppose that would depend on competing specificity constants between the two enzymes. Also, do you know if endogenous methylases would interfere with the protective value of introducing the complementary methylase to the restriction enzyme? I am none too familiar with the details of methylation patterns.
Thanks for the info, Derek. The purification is probably the hardest bit, because ideally it would have to be something easy for a DIYer to set up in their home lab (which would be the whole point of this endeavor). Perhaps it could use something like a polyhistidine tag for purification and maybe a GFP moiety for visual confirmation. I would have to look into the ease of home brew his-tag purification, though. I would also have to check up on any patent restrictions involving those. The thing with using commercial enzymes as a DNA source is an interesting idea. I may look into that for personal use, but that is definitely not viable for open-source enzymes.
On Thursday, March 8, 2012 7:45:15 PM UTC-5, Darrell Montana wrote:
-- On Thursday, March 8, 2012 7:45:15 PM UTC-5, Darrell Montana wrote:
Just a heads up on making restrictions enzymes... If you are going to try this you need to co-express the complementary methylase. If not, the RE will chop up the coli genome and kill the host...On Thu, Mar 8, 2012 at 11:45 AM, Derek <derekja@gmail.com> wrote:We started to put together such a project up here at the University of
Victoria for an igem team, but ultimately decided to do something else
because of the difficulty of the purification problem. Taq is pretty
easy since it is thermostable, but restriction enzymes need to be
purified in some way. Some of the earlier restriction enzymes are off
patent now, so depending on what you want that may not be as much of
an issue.
BTW. not an entirely ethical way of getting your plasmid (and one that
would likely hit patent restrictions), but one that sometimes works,
is to take a commercial enzyme and use it as the source of
transformation DNA. Make sure you use high competency cells for this
because there's not much DNA there, but as good as the commercial
purification strategies are they often leave enough plasmid DNA in the
mix to transform with. You still need to figure out what antibiotic
they used and reverse-engineer the purification strategy.
--Derek
On Mar 8, 11:11 am, Ethan <argentu...@gmail.com> wrote:
> Forgive me if this has already been discussed, but what is the prevailing
> opinion on open-source enzymes. For doing DIY plasmid engineering, the most
> costly part is probably the restriction enzymes and ligase. Any kind of
> genetic manipulation (PCR, etc) gets expensive as a result of enzyme
> prices. Do you think it would be possible to develop some sort of
> open-source production method for useful enzymes (ie. open source vectors
> for expressing the enzymes and some easy way to purify them)? This is all
> just speculation, and my research into the subject has been limited. My
> experience in protein purification is also quite narrow.
>
> I am aware that Cathal has an open-source plasmid vector in the works. If
> there is an effort to create open restriction enzymes, it would be
> convenient to coordinate them with the restriction sites on the plasmid. I
> am just looking for input on all aspects of such a project. What are the
> legal restrictions with gene patents? Do you think that it is viable to
> have some sort of chromatography set up that is reasonable for a large
> number of DIYers to have access too for purifying the proteins perhaps via
> a common tag? What do you think the costs (and benefits) of an endeavor
> like this might be? Thanks for your thoughts!
>
> -Ethan
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