The nickel isn't just encapsulated, it's held by a special chelating agent that's covalently bound to the agarose beads. Very hard to DIY I imagine...
Nathan McCorkle <nmz787@gmail.com> wrote:
>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 som
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