Is there interest in lower cost restriction enzyme varieties?

[BioGuy]

Hi everyone, I'm just trying to get a feel for who out there in DIYBio land might be interested in something like this. As it is right now the cost of restriction enzymes is anywhere between $55 - $200 per 10,000 units. I was thinking if your an independent scientist or working in a community lab 10,000 units might be a bit of overkill and would last an extremely long time. So I'm trying to see if there is interest out there for an option to purchase 500 - 1000 units of restriction enzymes for about $10? My thinking is that while it would be higher cost/unit the cost of having a variety of restriction enzymes would be immensely reduced.

[SC]

Hi Bioguy,

Your idea is a good one for local buyers, for example a community freezer program.  I'm not sure it would work as well for remote users.  REs must be shipped on dry ice, and with regular orders the cost of shipping is almost as much as the enzyme.  For a small order as you suggest, the cost of shipping would be more than the enzyme.  Personally, when I have to pay for dry ice shipping, I tend to stock up. 

 

I do like the idea of users being able to buy small amounts of things though.   Maybe some other type of supply that could be shipped without freezing would work better?

 

Stacy

 

[Ilya Levantis]

Well the idea would be to enable someone to order a set of 10 various enzymes (smaller quantities) at the same price of buying larger quantities of only 2 or 3. Shipping would therefore have the same relative cost per order as before.

[SC]

Ah, I see.  Yes, that would be useful: a variety pack of smaller quantities of enzymes.

[Mega [Andreas Stuermer]]

There is that paper that says some restriction enzymes are stable at room temperature for 6 months - and still retain like 99% activity after that period IIRC. 

I don't remember which enzymes it were (BamHI?)... The paper was discussed here a year ago or so...

If you kept them at 4°C or -20°C  that would surely be better. And available. 

[Koeng]

This makes sense. Some enzymes are more resistant than others though. BbsI, for example, begins to degrade at -20. 

I would like to know if there is a relationship between protein stability at room temperature and heat inactivation... I'd assume so since its all about protein stability. BamHI can't be heat inactivated (super unfortunate for BglBrick assembly, however I am actually currently discussing with NEB about one of their variants that is heat inactivatable! Gotta have BglII too though).

It seems like another simple experiment could be in place here. Keep a stock of diluted BamHI at room temperature and a stock of diluted BamHI at -20. (dilution because enzymes are expensive man). Keep these both at their temperatures for a month. After that month, take them both out and do serial dilutions and run it on some lambda DNA, then do a gel. The dilutions will eventually be below a unit, and then not cut all of the lambda DNA. In a perfect world, this would be at the same dilutions of both. In a realistic world, I would estimate a ~75% loss of activity because I am a pessimist.

If I get time next week I'll set up that experiment

btw, sorry for changing the topic on the thread. Really if you want good enzymes, just work to buy em from NEB. I talked to one of the price specialists a week or 2 ago and the main reason they do the quantity is because they want a universal base price of 'around 50 dollars for 500 units' universally. Most labs only use a few enzymes anyway, and a variety pack wouldn't really be useful to *most* labs because there isn't a need. They already have their cloning systems down and know what to use. If enough DIYbiologists get together to actually ask, however, and show there is a demand for it, I don't see any reason why they wouldn't go for it, other than it is hard to do that little of enzyme in a tube. If it comes to expressing it yourself, I have SpeI and XbaI fully synthesized with their methylase, you just need to actually clone it into an expression vector. Purification is a whole other issue, one I'm not skilled at :) . I honestly think it is hard to beat NEBs prices

-Koeng 

[Brian Degger]

https://www.neb.com/products/e0546-biobrick-assembly-kit is avalioable
for biobrick assembly.
Not exactly what you are wanting but interesting, if a little pricey still.

The BioBrick® Assembly Kit provides a streamlined method for assembly
of BioBrick parts into multi-component genetic systems. BioBrick parts
are DNA sequences that encode a defined biological function and can be
readily assembled with any other BioBrick part. The process for
assembling any two BioBrick parts is identical and results in a new
composite BioBrick part.

The BioBrick Assembly Kit contains EcoRI-HF™, XbaI, SpeI, PstI, T4 DNA
Ligase and NEBuffer 2.1.

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http://makerspace.org.uk
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[Josiah Zayner]

I think shipping is a big issue.
I run a DIYBio store, The ODIN (http://www.the-odin.com), and I have purchased and shipped many "thermostable" enzymes and tested them. There is a marked decrease in activity after shipping. Depends what you are trying to do but usually this is not a good thing.

If people know the quality of enzyme they are going to receive is not great then it might work. Most restriction enzymes at NEB and such are not really cost prohibitive these days at ~$50-$100 USD. Especially if you do experiments at a community lab and can find someone to split it with you. I usually stick with two enzymes for all my cloning(NdeI and BamHI) this saves alot of time effort and money.

Of course these days there are also many methods that are restriction enzyme free for cloning.

[BioGuy]

Basically what I'm trying to brainstorm right now is how can we continue to make independent biology more accessible. Barriers to entry have been greatly reduced in terms of equipment used - with various open source PCR designs, DIY PCR projects, DIY gel electrophoresis, etc. However not much has been done to reduce cost/barriers in acquiring reagents. I think a good first step might be making restriction enzymes more accessible.  Preferably to someone that doesn't have an advanced degree - think students in high school, aspiring amateur biologists, tinkerers, hobbyists, etc. 

I suppose what I want to see is people tinkering inexpensively in their garages similar to what people did in the 70s with electronics and computers. I want to see people be able to teach themselves hands on, in their own way, and on their own time about biotechnology. Were not there yet but I hope soon we can target consumers and tinkering can be as easy as purchasing a kit for biotechnology similar to these for electronics http://www.radioshack.com/electronics-learning-lab-kit/2800055.html#.VGrck3Wx15Q or like this for learning about microcontrollers http://arduino.cc/en/Main/ArduinoStarterKit. If you are tinkering with various plasmids, inserts, or want to ligate multiple inserts it seems to me you will quickly find yourself needing more than just a few restriction enzymes. But lets say you had 5 different varieties of restriction enzymes. This would most likely cost you around $275-$300 (assuming you buy the smallest quantities available) + shipping costs. For a hobbyist or tinkerer, who has around $100-$300 per month to spend on a hobby, then otherwise very intelligent people will probably simply take the path of least resistance and teach themselves how to program a computer, how to build computers, how to tinker with electronics, etc. 

As it is right now we don't live in a world where exploring and tinkering with biology is accessible. Even though it looks like there are quite a few community labs around the world (http://diybio.org/local/) there are still very few relative to the amount of people who may be interested in biology and geographic distances still make them quite innacessible for most. Plus there are still plenty of people who would prefer to tinker in their own home or workshop. I think both community spaces and independent spaces have advantages. I think one benefit of being able to work in an independent space is not being affected by group think mentality in terms of what you 'should' or 'need' to learn. 

Lets imagine for a moment though that the price of chemicals and reagents wasn't an issue for most people, which I do think it is. To order a few reagents you still need to establish a commercial address, establish a business, or a non-profit that focuses on education or research and development. Imagine if you had to do this simply to acquire a few resistors, diodes, and ICs in the 1970s and 80s. You are required to do this even if you just want to order a simple educational kit from Carolina Scientific.

http://www.amazon.com/Python-Programming-Introduction-Computer-Science/dp/1590282418/ref=sr_1_3?s=books&ie=UTF8&qid=1416291834&sr=1-3&keywords=programming Right now the average person can purchase this book and accomplish everything described in the book. Though I feel that with a book like this http://www.amazon.com/exec/obidos/tg/detail/-/0805330402/biolink-20 the average person wouldnt be able to accomplish anything.

I think you all bring up an important point when it comes to the logistics of shipping.

@SC you gave me an idea. I wonder if any of the community labs out there would have interest in selling small quantities of restriction enzymes (and other reagents) to members and non members who may be a few hours drive away - or simply don't desire to participate in a community lab environment. This could be a potential source of revenue for community labs, reduce costs of membership, and further expand the boundaries of who can participate in independent biology exploration.

@llya Levantis - Exactly! But after reading the other comments I realized there will most likely be a logistics issue with individuals needing to restock only one of the restriction enzymes they are out of. Then there will be the logistics cost of shipping for single reagents.

I did a little searching for dry ice and it really doesn't seem that expensive. Seems to be in the range of $1-$2/pound. I believe thats a little over 1/2 liter. Too tired to do the math right now. Of course in the end I'll need to look into more what the costs/restrictions are with shipping dry ice/biological reagents, how quickly dry ice sublimates, etc.

I'm tired but want to think about all this more. Perhaps there wouldn't be much interest for people who are already involved with DIYBio - and if I really want to make things more accessible for people learning from the ground up perhaps restriction enzymes are not the best way to start - as you can't exactly do anything with just restriction enzymes. So perhaps I should focus more on something like an inexpensive educational kit. I wish there were a way to benefit both communities - those with education / or who have access to a community labs, and those without. Perhaps pursuing an idea to see if community labs also would want to serve as distribution hubs might be worthwile I'm not sure. If community labs did have interest in this then it might be possible in the future for individuals to purchase a book on biology and biotechnology experiments, while purchasing what they need from community labs.

@Josiah Zayner I really like what you've done. I actually didn't think there were any sites/online stores in existance like yours. Need to take a closer look later.

[John Griessen]

On 11/18/2014 01:35 AM, BioGuy wrote:
> you all bring up an important point when it comes to the logistics of shipping.

Perhaps a first step is a low cost dry ice shipping box with a super insulated
dry ice frozen compartment, a fan, and a fridge compartment. Without any
control, dry ice shipping is freeze-thaw, then not necessarily come to life.

My proposed shipper-frigerator would maintain a 4 deg C temperature for many days
of shipping. The size could be made to fit inside flat rate boxes
so they could be used when full of dry ice at a $13 rate,
then for dead-head return shipping of empty ice boxes
the under 13 oz first class parcel rate would be under $5.

An ice box version with a slightly more permanent exterior would do duty
in a lab and would work with water ice cubes and a drain bucket,
or with dry ice.

[Koeng]

I’ll give my 2 cents here- there’s a reason reagent cost isn’t going down. These things are very hard to produce, an with restriction enzymes, NEB practically has a monopoly because of their quality (Not that they’re abusing it, they’re prices are often lower than other companies, combine that with their quality testing its worth it). Protein purification, as Cathal learned from his attempt at a DIY purification method, is pretty darn tricky. A lot of work goes into optimization of purification of the common enzymes, plus quality checking for activity takes even more. Much of the work comes from unpaid interns. At the current time, I do not think restriction enzymes are prohibitive to DIYbiology in a school or small hobbyist setting. NEB has sent restriction enzymes for free to my school when I needed it for teaching there, and small hobbyists can go ahead and pool to buy them. (I only talk about NEB here because they’re the only people I have experience with, good experience too. Highly recommended)

I think that DNA, not enzymes, are the prohibitive. Enzymes are cheap enough*, DNA is not. I’ll go with your point of 5 different restriction enzymes costing a lot… but how much of them are you going to really use? I work at a college level lab and we very very rarely run out of enzyme, a lot of the time we actually stop using some tubes because they get so old! 

There is one time where restriction enzymes are prohibitive though: when you are cloning and working with plasmids from a variety of labs. Each lab has their own favorite enzyme to use for certain things, and so that leads to needing a HUGE amount of enzymes because PCR is usually difficult (mutations).

How do you fix this? Standardize the plasmids of course! With a 250$ kit from NEB you can begin combining biobrick parts 50 times! (https://www.neb.com/products/e0546-biobrick-assembly-kit). Thats only 5$ a reaction, which is a reasonable price if you look at how much it normally costs to clone. Overall, this is a nice way to get a good introduction.

“That’s great”, now people can tinker around with biobrick plasmids. But even this isn’t true. The biobrick kit requires you have an iGem team, which is a whooping 3500$! (Shoutout to everyone that went to the iGem jamboree, it was fantastic). But 3.5k isn’t a good price for anyone, its a bit pricey. Plus they send you the entire registry, which is over a thousand plasmids! Pretty much no one uses all of them. Actually, there is a limited amount of parts people actually use over and over, including a few RBSs and a few promoters and a few proteins. They really need ***TO PROVIDE AN AFFORDABLE BIOBRICK KIT***. 20 plasmids is EVERYTHING needed for the starter DIYbiologist. Actually, I think you could reduce that number to just 10. That’s all that would be needed for a beginner DIY biologist. A few operators, few RBSs, few promoters, few terminators, few proteins (repressor and GFP). That’s ALL that’s needed for a beginner. That’s pretty much all I’d need at the current time with their kit to make some of my own constructs for teaching people. Sell that for 200~$ and I think it would be affordable enough. 500~$ for 20 plasmids and the enzymes needed for manipulation is a fair price, I spent that much on enzymes alone when I began. If there’s anyone on the iGem board reading this, I would love to see this happen!^ or just give me permission to distribute it and I’ll offer it myself because I also want to see independent labs popping up, and also want to get to know them :)

-Ko

[SC]

Justin,

Maybe I misunderstood your intent.  Were you planning on purifying your own enzymes or repackaging larger quantities from NEB into smaller aliquots?

Stacy

[Dakota Hamill]

I've met with NEB about this same thing I go there once a month.   their system has been perfected over 30 years.  They do sell a lab starter pack with shrimp phosphatase, restriction enzymes,  polymerase, ligase, etc.  I'm on my phone and I can't find it.    I'm not saying it's a bad idea because hell, I want to do the same thing.  I came to realize I don't think the market is there.  Sure maybe a few people on the list might help out or you could find some high schools, but no real lab is going to drop NEB for a cheaper enzyme seller who can't disclose they buy from NEB anyway.

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[Cathal Garvey]

This is why I was going to get into the "grow your own enzymes" market
instead with Glowbiotics. Making and selling purified, certifiable
enzymes is the sort of task that's economic-at-scale, which encourages a
natural monopoly to the largest and best established player.

There are plenty of groups selling ultra-cheap EcoRI, and some large
competition with NEB who go for quality over value, there's very little
room for a successful small player.

Instead, I thought there was room for a seller of grow-your-own kits,
starting with basic plasmids and working up to enzyme-production systems
that used trivial purification methods. Those who've seen me exposing
these at Synbio Future (http://www.indiebiotech.com/?p=245) will be
familiar with the Agar-based method I tried, but failed, to get to work
(let that count as prior art towards the idea of "using agar/ose/opectin
as a purification matrix". I still think this method could work but the
domain I was trying to use didn't play well with others.

It didn't turn out well for my plasmid plans, but it's possible that if
someone can work out a stack consisting of:
* Universally applicable, cheap, trivial purification (Is MBP out of
patent yet? Use with hot-filtered corn-starch to get MBP-bindable starch?).
* Universally applicable inducible expression standard, for production
of enzyme efficiently in culture.
* A library of well-characterised expression constructs including
preferred incubation conditions, perhaps leaning towards antha-lang.org
and OpenTrons for automated growth/induction/purification

..I think there'd be a modest market for that. But "modest" is key, I
made the mistake of overestimating this market in the past.

On 21/11/14 00:23, Dakota Hamill wrote:
> I've met with NEB about this same thing I go there once a month. their
> system has been perfected over 30 years. They do sell a lab starter
> pack with shrimp phosphatase, restriction enzymes, polymerase, ligase,
> etc. I'm on my phone and I can't find it. I'm not saying it's a bad
> idea because hell, I want to do the same thing. I came to realize I
> don't think the market is there. Sure maybe a few people on the list
> might help out or you could find some high schools, but no real lab is
> going to drop NEB for a cheaper enzyme seller who can't disclose they
> buy from NEB anyway.
>
> On Nov 20, 2014 4:47 PM, "'SC' via DIYbio" <diy...@googlegroups.com

> <mailto:diy...@googlegroups.com>> wrote:
>
> Justin,
>
> Maybe I misunderstood your intent. Were you planning on purifying
> your own enzymes or repackaging larger quantities from NEB into
> smaller aliquots?
>
> Stacy
>
>
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[SC]

I use a small to moderate amount of enzymes, and I always buy them from NEB.  I do not enjoy the thought of purifying my own enzymes.  It seems i would spend a lot of time and energy only to end up with a potentially less pure product that would mess up my downstream workflow just to save a few bucks.  If the goal for hobbyists is to see if you can make the enzyme and practice protein purification, then I would see value in such a kit.   if the goal is to get a reliable product to produce something else, and the rest of the workflow is expensive and time consuming, I wouldn't risk it.

 

What may be a more workable, and profitable, venture is to set up an NEB freezer program in your area, assuming it needs one and you have potential customers. It would be on the up-and-up with NEB and personally, when I need an enzyme, I'd rather drive down the street and shell out $30 and come home with the product then order it from NEB, pay shipping, and wait.  

 

My two cents.  

[Jeswin]

Aren't there Chinese companies that sell enzymes? I remember coming
across some a few years ago. I'm not sure about the prices though.

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[Nathan McCorkle]

On Fri, Nov 21, 2014 at 5:09 AM, 'SC' via DIYbio
<diy...@googlegroups.com> wrote:

> What may be a more workable, and profitable, venture is to set up an NEB
> freezer program in your area, assuming it needs one and you have potential
> customers. It would be on the up-and-up with NEB and personally, when I need
> an enzyme, I'd rather drive down the street and shell out $30 and come home
> with the product then order it from NEB, pay shipping, and wait.

Unless their business setup is adjustable, my last experience with an
NEB freezer at the University that was up the road from my University
ended with the other University saying the freezer was only for people
who were in labs over there. No matter that NEB would ship me enzymes
at my school just fine, and would likely save on shipping overall to
with me driving up the road instead. Whether this practice was at the
discretion of the University or NEB, I don't know, they made no
distinction.

[lareine]

We sell high quality restriction enzymes at the fraction of the NEB price: http://www.aasinc.co/store-and-promotions.html#!/c/0/offset=0&sort=normal

[Sebastian Cocioba]

I can has NcoI? o.O

These prices are awesome! I'm a huge fan of traditional cloning and wish more people would cut the whole expensive gibson, splice, goldengate bs and just learn using reliable dual cutter cloning. Its like saying "I want to learn to drive a car but first I must purchase a Bentley". Surprised you don't carry EcoRI and the other biobrick standards as they are also super handy for diagnostic cutting post mini-prep and should be cheap as all hell. I use EcoRI daily for routine diagnostic cuts of my supposed clones and NEB has me by the...belt...with costs. I would be happy to review the parts for you for some kind of discount if you would like. I literally only do restriction cloning in my work. It may be slow but its reliable as hell and that's very hard to come by nowadays...

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[Koeng]

That's surprising, I have actually gotten more reliable results from SLiCE and gibson than restriction enzyme cloning (even when I have single part cloning I do those)... I guess it's just up to what you're used to.

Are you planning on adding any new enzymes, like EcoRI and SpeI, anytime soon? Those are my most commonly used and it would be worth to start getting those if so because then I would have a complete biobrick series from a single provider (for cheaper)

-Koeng