polymerase expr/purification WAS:Anyone need some synthesis?

[Jeswin]

On Fri, Mar 21, 2014 at 5:25 PM, Koeng <koen...@gmail.com> wrote:
>
> Hey
>
> I am doing a large synthesis order soon from gen9. However, they insist upon synthesis of at least 25kbs, and I unfortunately only need 20kbps synthesized. If anyone wants to get some genes for cheap, contact me. It is 20 cents a base pair ( .5 to 3kbp) and as a little incentive I will also include a (native) pfu polymerase gene for expression in E coli :)
>

How hard is it to express and purify polymerase in E coli? I was
browsing Engelke's paper on it. It looks complicated and not easily
done unless you got some specific equipment. Otherwise, it's very
doable. Anyone here tried this?

Engelke, David R., et al. "Purification of Thermus aquaticus DNA
polymerase expressed in Escherichia coli." Analytical Biochemistry
191.2 (1990): 396-400.
Available at: http://deepblue.lib.umich.edu/bitstream/handle/2027.42/28292/0000046.pdf

[Dakota Hamill]

openbiotech.org or something had a kit for that I believe. master
mixes from NEB are so good and cheap though

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[Sebastian Cocioba]

Did you see the protocol from Openbiotech website? They sell their
pOpenTaq vector and explain how to purify. Seems straight forward
albeit tedious.

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D From: Jeswin
Sent: 3/21/2014 6:39 PM
To: diy...@googlegroups.com
Subject: [DIYbio] polymerase expr/purification WAS:Anyone need some
synthesis?

[Nathan McCorkle]

that extraction looks pretty straightforward, most of those ingredients can likely be substituted (its just grow, centrifuge, rinse, rinse with lysozyme and detergent, purify with ion-exchange), the PEI resin isn't terribly expensive, but seems like there should be a cheaper substitute... maybe just any ion-exchange resin... Cathal?

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[Mega [Andreas Stuermer]]

His-tag, but that is patented IIRC. Tjat would be easy to purify, just pour over a nickel column.

[Mega [Andreas Stuermer]]

[Koeng]

It was just my idea to do pfu because look at how cheap they can make taq... it would be essentially the same protocol

and NEB's stuff is like a dollar a PCR

[Sebastian Cocioba]

I know u hate taq but did u see the eBay offer of $23/1000u? And I believe 50,000u for $200. Thats cheap. SydLabs has a one time deal of 20% off an already cheap price too.

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D

---

From: Koeng
Sent: 3/21/2014 8:19 PM
To: diy...@googlegroups.com
Subject: [DIYbio] Re: polymerase expr/purification WAS:Anyone need some synthesis?

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[Cathal Garvey]

Taq's error rate is an issue, but adding a small amount of proofreading
polymerase can make up for that. So you taq's speed and affordability,
and error-correction/processiveness provided by other pols.

I think the apparent difficulty in the early papers can probably be
heavily attributed to poorly optimised DNA; Thermus aquaticus is hardly
a close relative of E.coli. I haven't looked at the codon usage tables,
but I wouldn't be surprised to see an issue there.

Same goes for most thermophiles, so I'd caution against using them
without even a little optimisation? :)

Also, watch out for inteins, I recall finding a proofreading thermopol
in uniprot that was *huge* but a large portion of that was an intein, so
could be dispensed with when designing an artificial gene if you
identify the intein splice sites and chop everything between them out.

That also introduced me to the existence of inteins, which are bonkers.

On 22/03/14 00:24, Sebastian Cocioba wrote:
> I know u hate taq but did u see the eBay offer of $23/1000u? And I
> believe 50,000u for $200. Thats cheap. SydLabs has a one time deal of 20%
> off an already cheap price too.
>
> Sebastian S. Cocioba
> CEO & Founder
> New York Botanics, LLC
> Plant Biotech R&D

> ------------------------------
> From: Koeng <koen...@gmail.com>

> Sent: 3/21/2014 8:19 PM
> To: diy...@googlegroups.com
> Subject: [DIYbio] Re: polymerase expr/purification WAS:Anyone need some
> synthesis?
>
> It was just my idea to do pfu because look at how cheap they can make
> taq... it would be essentially the same protocol
>
> and NEB's stuff is like a dollar a PCR
>
> On Friday, March 21, 2014 3:39:09 PM UTC-7, phillyj wrote:
>>

>> On Fri, Mar 21, 2014 at 5:25 PM, Koeng <koen...@gmail.com <javascript:>>

> https://groups.google.com/d/msgid/diybio/eecf777a-c6a4-4811-8d89-ab902fadd473%40googlegroups.com<https://groups.google.com/d/msgid/diybio/eecf777a-c6a4-4811-8d89-ab902fadd473%40googlegroups.com?utm_medium=email&utm_source=footer>
> .

> For more options, visit https://groups.google.com/d/optout.
>

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[Koeng]

Yep that was the idea. Error correction. A lot of the time I need to do HUGE PCRs which taq is not good at (So many errors) in which case I use Q5. 

By the way, anyone know what Q5 polymerase is? Its like over 100x less error prone then Taq and like 3x as fast. But I couldn't find an organism they got it from... and when I looked at their patents last (it was a while ago) it had something on DNA binding domains. Might be a synthetic polymerase

Back on topic, pfu didn't seem to have any and was a decent size, only like 2300bp or something. Intiens... those sound interesting. (It might have some relevance to another project I am doing, thanks for bringing it up!)

[Josiah Zayner]

Unless one is amplifying >1.5kb Taq should not be an issue. Though people remark on Taq polymerases "high error rate" when cloning this is often not an issue because at the end of the experiment one is only using a single copy of the gene to insert into a plasmid. Sequence two clones and as long as you did everything correct your chances should be very high that one or both of them contain the correct sequence.

His-tagged polymerases are very easy to purify if you have access to Nickel.Metal affinity column. Many labs purify their own Taq for things like genotyping that require sooo much polymerase, hundreds of reactions a week. But otherwise it is like trying to make a cheaper hamburger than McDonalds, you will not come close to price matching unless you purchase in large bulk.

To me time is money and to most labs time is money and purification takes time and effort and usually there is no QA involved.

Thermo sells Phusion for $82/100 rxns which is a master mix
http://www.thermoscientificbio.com/pcr-enzymes-master-mixes-and-reagents/phusion-flash-high-fidelity-pcr-master-mix/

Buying dNTPs for ~200 rxns is $30 alone
http://www.thermoscientificbio.com/general-reagents-and-accessories/dntp-mixes/


So let's say one liter of bacteria gives you enough protein at the end for 1000 rxns which would be $820
Gene synthesis: let's say it's only around $200 for arguments sake(pfu is ~900AA? so thats >2000bp more like thousands of $$)
Cloning? (Restriction Enzymes, &c.) Add what you think is reasonable
~$100-150 is the cost of the dNTPs for 1000rxns
Let's say reagents and stuff for expression.lysis.storage $150
A day or two or three or seven or 20 of work optimizing the protocol so your expression and purification work ~$150(let's keep it cheap because this is DIY) the verifying that the polymerase works figuring out how much polymerase needs to be added for each batch, verifying the purity of your enzyme, removing an DNAse contamination, &c.
Nickel or Ion exchange column($50-$100 probably much more but let's keep it cheap)

Your cost is at least $700 even if it was only $400 or $300. Think about that you won't begin to make your money back until you run close to 1000 rxns and even then it's not like you are saving $$$$ you are only saving $. And most likely your cost is going to be in the couple thousand dollar range

For $400 USD you can run about 500 PCR reactions. If you are running 500 PCR reactions either something is wrong and your protocols are failing or you are using consumables at such a high rate that you need to find $$$$ in funding. I think for my whole Ph.D. thesis I ran about 500-1000 PCR reactions over 5 years and cloned 200-400 constructs.

As Sebastian said enzymes are cheap. And they will probably only become cheaper. But I am sure other people feel different.



Josiah

[shamrock]

In keeping with the DIY ethos at BUGSS we’ve put together a kit for expressing and purifying your own Taq. The kit contains the construct, expression strain, inducer, and buffers for doing the purification. From a 1 ml culture you can purify enough Taq for 20-50 PCR reactions. Purifying Taq from other proteins is pretty simple and you can get usable Taq with a simple heat precipitation and centrifugation-getting rid of DNA contamination is a bit trickier - even commercial preparations are often contaminated with bacterial DNA. With the BUGSS kit you’ll be able to do close to 200 – 1 ml expression/purification operations and since you’ll get enough Taq per purification to do 20-50 PCR reactions were talking pennies per reaction. Of course there is some time involved and you do need dNTP’s (were working on that part as well)and primers but the cost of the enzyme will no longer be an obstacle to doing all the PCR you want.

We’ll be announcing the BUGSS store in a couple of weeks. We’ve got a few other things that people in the DIY community may be interested in as well including what we call our syn-bio starter kit. It’s nothing revolutionary just a collection of the solutions, strains, and reagents needed to do some simple DIY experiments and get your hands dirty.

Keep an eye on the BUGSS web site (www.bugssonline.org) for the opening and I’ll announce it here as well.

[Koeng]

Sounds awesome! Would there anyway to port the kit for use with pfu polymerase (since it is a better polymerase and you produce it yourself so there isn't any reason not to use it except speed)

Like what expression strains/ inducer will you be using? I could probably get it synthesized with the needed promoter/operator/RBS