Open-Source Enzymes

[Ethan]

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

[Derek]

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

[Darrell Montana]

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...  

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[Ethan]

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.

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[Darrell Montana]

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

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[Ethan]

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.

[Nathan McCorkle]

On Sat, Mar 10, 2012 at 3:46 PM, Ethan <argen...@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

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--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[Cory Tobin]

> That and the fact that navigating the US patent office website
> is, like almost everything to do with our government, a complete nightmare.

There's an easier way to find which enzymes are patented. NEB has
this website called REBASE http://rebase.neb.com/rebase/rebase.html
You can find restriction enzymes using the second search box. Lets
say you are looking for EcoRI, and end up here
http://rebase.neb.com/rebase/enz/EcoRI.html Over on the right you can
find links to the protein and nucleotide sequences, which would be
useful if you wanted to synthesize the enzyme sequences. Also on that
page is a link to related references. If the enzyme is patented it
will be listed on there. There's often hundreds of references, so
just Ctrl+F to find patents. And just FYI, there's often multiple
patents on any one enzyme (relating to different applications of
embodiments of that enzyme) so you may have to read a few of them to
find which one pertains directly to your intended application.

-cory

[Cathal Garvey]

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 <argen...@gmail.com> wrote:

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[Cathal Garvey]

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 <nmz...@gmail.com> wrote:

[Mac Cowell]

Would ice-affinity purification work? You link an anti-freeze protein moiety to your protein of interest, express, lyse, and expose the lysate to a probe that is slowly and evenly cooled (?) to encourage ice nucleation.  At some point afterwards the AFP is cleaved off.

Overview: http://2011.igem.org/Team:Yale/Protein

The take team has a poster with more info.

Mac

231.313.9062 // @100ideas // sent from my rotary phone

[Cathal Garvey]

When I looked up ice nucleation proteins on google patents I found some worrying looking patents. Universities don't usually worry about patents, so I'm not sure how seriously to take the implied suggestion that ice proteins can be "Used by anyone". Still, would value more expert opinion there: are ice proteins unencumbered by patents?

Mac Cowell <m...@diybio.org> wrote:

--

[Serhat Sevli]

When I looked via Google

"restriction enzyme patent expire"

there are lots of production methods that are expired.

is it already forbidden to produce by other companies or DIY

11 Mart 2012 01:16 tarihinde Cathal Garvey <cathal...@gmail.com> yazdı:

--

Serhat Sevli, MSc

Genetikçi (Geneticist)

+90-535-628-7887
erke....@gmail.com

[Darrell Montana]

Serhat -

You can also search a uspt.gov..   (link here http://patft.uspto.gov/netahtml/PTO/search-adv.htm)

In the box use the query below:

ABST/(restriction AND endonuclease)

You will get a list of all US patents that contain restriction and endonuclease in the abstract.   Look for the dates the patents were filed.  All patents filed 20 years ago or longer are now expired and anyone is free to reproduce the work.  Below is a patent from NEB for production of the very useful restriction enzyme NdeI.   The patent expired in 2008.  Good luck

http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.htm&r=252&f=G&l=50&d=PTXT&s1=%28restriction+AND+endonuclease%29.ABTX.&p=6&OS=abst/%28restriction+and+endonuclease%29&RS=ABST/%28restriction+AND+endonuclease%29

[Cathal Garvey]

What's the rule here: Does a patent expire 20 years from date of issue,
or of application? Many patents are applied for years before they are
published, so an "Invention"* may be have been issued a patent 5 years
after application.

Don't complain about searching US patents, btw. You guys have it
insanely easy compared to the EU patent office(s!!!). You have Google
Patents, Patentstorm and heaps of other organisations to make your
searching easy.. we have one atrocious clearing-house with awful, awful
software, often without any supporting documentation besides the title
of a patent, and to get any real information you're then directed to
individual country patent offices.

Oh, and EU patents are often applied for or granted years after the US
equivalents, apparently without backdating to the supposed time of
"Invention"*.

*Where "Invention" means "DNA I was luckily the first to read, and stole
from you all for two decades, kthx".

On 12/03/12 11:57, Darrell Montana wrote:
> Serhat -
>
> You can also search a uspt.gov.. (link here
> http://patft.uspto.gov/netahtml/PTO/search-adv.htm)
>
> In the box use the query below:
>

> *ABST/(restriction AND endonuclease)*

>> *Serhat Sevli, MSc*
>> *Genetikçi (Geneticist)
>>
>> +90-535-628-7887
>> erke....@gmail.com*

>>
>>
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>

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[Darrell Montana]

Hi Cathal -

In the US, the patents expire 20 years from the date they were filed.  (http://en.wikipedia.org/wiki/Term_of_patent_in_the_United_States). 

A good site to browse international patents is http://www.wipo.int/patentscope/search/en/search.jsf