On Sat, Mar 10, 2012 at 3:46 PM, Ethan <argentumxy@gmail.com> wrote:
> The scale of this project is rapidly increasing from what I anticipated. I
> suppose that if the genetic sequence is in the public domain, then there
> shouldn't be much problem with pulling it straight out of a commercial RE,
> but I would have to look into it. I am just very wary of the legal
> restrictions because I would not like to end up (as a poor student) being
> sued by some mega-corporation over a patent or some legal nuance that I
> overlooked. That and the fact that navigating the US patent office website
> is, like almost everything to do with our government, a complete nightmare.
>
> As of now, this is all purely hypothetical discussion. I am going to be away
> from my lab while at school, and I don't currently have the time to sink
> into such a project. That said, I would be very interested in going after
> this in the future. You raise a good point about the online-funding thing
> (like the PloS ONE paper that was recently published with kickstarter
> funding from members here). That would reduce the financial problems of
> getting this going.
>
> The T7 system definitely looks like a good promotor to use for this, and I
> will keep it in mind. As for his-tag purification, it doesn't seem like ion
> exchange resins are as readily available as I would like for something like
> this. I am curious about finding an alternative method that could use items
> that pretty much anybody could access (maybe something along the lines of
> silica powder chemically modified with some sort of reagent that is
> available for purchase in stores). That would be ideal, but I don't know if
> it is the least bit viable.
His columns are Nickel based, usually suspended in agarose beads...
they're rechargable for a while until the agarose falls apart enough
and the Nickel is in solution with no way to precipitate out (the mass
of the agarose bead)
Maybe these new-age cooking techniques (molecular gastronomy) could be
used to make DIY Nickel beads:
http://cookingissues.wordpress.com/2009/05/18/agar-tobiko/
http://willpowder.net/sodiumAlginate.html
http://www.eatfoo.com/archives/2007/12/recipes_more_spherification_wi.php
the agar gels completely unlike the alginate technique
http://michaellaiskonis.typepad.com/main/files/raspberry_pearls.pdf
>
>
> On Saturday, March 10, 2012 9:16:25 AM UTC-5, Darrell Montana wrote:
>>
>> Hi Ethan -
>>
>> You bring up many good points open-source enzymes.. Do you really think
>> using REs as a source of DNA would be a problem for making open-source
>> enzymes? I have never read the fine print of the agreement between a RE
>> supplier, but think it would be very easy or even legal, to enforce.
>> Another way to get at the enzyme is to find some sort of online-funding site
>> to raise the $150-$1000 you would need to have the coli expression
>> construct made synthetically (they typically cost ~$1.25 / amino acid, so
>> small 120 residue proteins will cost you ~$150 and large 500 residue enzymes
>> will be closer to $600) If you agreed to freely distribute the plasmid
>> and purified protein to anyone who "invested" I think you could have a lot
>> of takers, especially if the protein was for a common tool like EcoR1, Taq
>> Pol, BamHI, Ligase, etc etc.
>>
>> I think there is too much worry regarding IP rights for many of these
>> tools. While many are still "on-patent", many of them have long ago
>> expired, but are often still advertised as requiring a licence. A good
>> example is the T7 expression system that every supplier advertises as
>> "requiring a license from Brookhaven National Lab." The original patent
>> for the T7 system was filed on Oct 19, 1989, and thus expired in Oct 2009.
>> While I'm no lawyer, I think this means T7 systems are now fair game to
>> hackers..
>>
>> Here's a link to the patent
>>
>>
>> http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.htm&r=32&p=1&f=G&l=50&d=PTXT&S1=%28t7+AND+expression%29.ABTX.&OS=abst/%28t7+and+expression%29&RS=ABST/%28t7+AND+expression%29
>>
>> I think if you dig you will find that many common synthetic biology tools
>> like the T7 system...
>>
>>
>> If I were to make open source enzymes I would make His-tagged constructs
>> for easy purification from protein overexpressed in T7 systems. For many
>> enzymes and applications a simple batch purification using small amounts of
>> Ni-chelate beads would given enough protein to do a lot of work. For
>> example, from 100 ml of culture if LB it is reasonable to assume you will be
>> able to isolate 1mg of the desired protein. Since most synthetic biology
>> applications require ng amounts of enzyme, 1mg can go a long way. And I be
>> you would find a lot of beta testers for the purified material here in his
>> group.
>>
>> I think there would be many ways to successfully express the two genes. I
>> bet you could co-transform with two plasmids, or use a single vector to
>> express both simultaneously... You would have to do the experiments to find
>> out what was best on a case by case basis I think... Good DIY projects
>> though... There is a lot of literature on this.
>>
>> Good luck
>>
>>
>>
>>
>>
>>
>>
>>
>> On Fri, Mar 9, 2012 at 10:55 AM, Ethan <argentumxy@gmail.com> wrote:
>>>
>>> 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:
>>>>
>>>> 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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>>>>>
>>>>
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>>
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--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
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