Re: [DIYbio] Re: Open-Source Enzymes | Science

Accursed patents!

The issue of purification is one if the hardest questions to answer, consisting the stifling atmosphere of overbroad patents covering everything from maltose-binding to cellulose-binding to hair-binding peptides. Resins are basically no-go, as I doubt we can find an easy and benign crosslinking reaction for common polymers to create them.

Besides these engineering issues though, patents covering the enzymes are the next huge roadblock. By patenting the gene sequence, the companies were able to grab an effective patent on enzymes that had already been used for years by the research community. Worse, many more recent patents patent anything within a wide % identity score of the amino acid sequence, often so much that an unrelated distant orthologous protein could be considered patented as an unintended side effect.

It's insulting and stupid, and it means you risk a bearing if you simply use the sequences in the enzyme preps, if you make it obvious that you're doing so.

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.
>
>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 co
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Source: http://sciencenewstop.blogspot.com/2012/03/re-diybio-re-open-source-enzymes_5484.html

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