Fwd: search for plasmid containing Taq gene

[Cathal Garvey]

This, if true, is an amazingly handy tip for producing proteins as homebrews.

---------- Forwarded message ----------
From: DK <d...@no.email.thankstospam.net>
Date: 27 July 2010 16:35
Subject: Re: search for plasmid containing Taq gene
To: met...@magpie.bio.indiana.edu

In article <mailman.912.128023...@net.bio.net>, jh <bio...@gmail.com> wrote:
>Dear all,
>
>I write to search for a plasmid containing Taq DNA　polymerase gene.
>Anyone who have this plasmid would be kind to help me?
>Thanks in advance.

Have really highly competent cells and possibly try several Taq
suppliers: "transform" cells with the protein prep. Virtually every
recombinant protein prep is contaminated at a very low level with
the recombinant plasmid. Since it is not known what resistance
marker was used, plate on amp, kan and cam plates.

I have used this trick to get several genes.

DK

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[Nathan McCorkle]

I've heard this in a thread here before...

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[General Oya]

Our iGEM team is creating a BbPart for the TAq Polymerase, and it has been suggested that part of our testing and documentation utilize results from others. This may be our opportunity to share the part with the DIYBio community, without voiding any of our safety, security restrictions imposed by iGEM moderators.

Check out the project at http://2010.igem.org/Team:Baltimore_US/Project

If we are successful with the point mutation and construction, then we will attempt the ligase as well. Then perhaps a few restriction enzymes.

Tito do you know if this would be appropriate use of testing modalities?

Thanks,

Ryan Ogle, LMT
ad...@genoblasts.com

On Tue, Jul 27, 2010 at 5:10 PM, Cathal Garvey <cathal...@gmail.com> wrote:

--

[Russell Durrett]

Ryan -

Thanks for sharing your parts! I've thought of a couple of things to
do with Taq expression and will definitely use your biobrick one day.

If you're planning on expressing the biobrick restriction enzymes
(EcoRI, SpeI, XbaI and PstI) (and maybe NotI depending on your future
use) you would need to make sure that the enzymes do not cut the DNA
in the bacterial genome. If it does then the restriction enzymes will
probably be toxic or you will at least get low yield of enzyme.
Instead of using bacteria, you could use yeast to express the enzymes.
If you attach a yeast secretion tag (which was just biobricked by my
team @ NYU, we'll be submitting it soon or can send it straight to you
if you want) you could dump the enzymes into the ER.golgi secretion
system of Saccharomyces so they would never even interact with a
genome. This would also allow you to purify the enzymes straight from
the supernatant instead of from the cells.

Hope this helps with your project

On Jul 27, 6:25 pm, General Oya <general...@gmail.com> wrote:
> Our iGEM team is creating a BbPart for the TAq Polymerase, and it has been
> suggested that part of our testing and documentation utilize results from
> others. This may be our opportunity to share the part with the DIYBio
> community, without voiding any of our safety, security restrictions imposed
> by iGEM moderators.
>

> Check out the project athttp://2010.igem.org/Team:Baltimore_US/Project

>
> If we are successful with the point mutation and construction, then we will
> attempt the ligase as well. Then perhaps a few restriction enzymes.
>
> Tito do you know if this would be appropriate use of testing modalities?
>
> Thanks,
>
> Ryan Ogle, LMT
> ad...@genoblasts.com
>

> On Tue, Jul 27, 2010 at 5:10 PM, Cathal Garvey <cathalgar...@gmail.com>wrote:
>
> > This, if true, is an amazingly handy tip for producing proteins as
> > homebrews.
>
> > ---------- Forwarded message ----------
> > From: DK <d...@no.email.thankstospam.net>
> > Date: 27 July 2010 16:35
> > Subject: Re: search for plasmid containing Taq gene

> > To: meth...@magpie.bio.indiana.edu
>
> > In article <mailman.912.1280230802.25217.meth...@net.bio.net>, jh <

> > bio...@gmail.com> wrote:
> > >Dear all,
>
> > >I write to search for a plasmid containing Taq DNA polymerase gene.
> > >Anyone who have this plasmid would be kind to help me?
> > >Thanks in advance.
>
> > Have really highly competent cells and possibly try several Taq
> > suppliers: "transform" cells with the protein prep. Virtually every
> > recombinant protein prep is contaminated at a very low level with
> > the recombinant plasmid. Since it is not known what resistance
> > marker was used, plate on amp, kan and cam plates.
>
> > I have used this trick to get several genes.
>
> > DK
>
> > _______________________________________________
> > Methods mailing list

> > Meth...@net.bio.net

> >http://www.bio.net/biomail/listinfo/methods
>
> > --
> > letters.cunningprojects.com
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> > twitter.com/labsfromfabs
>
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> > diybio+un...@googlegroups.com<diybio%2Bunsu...@googlegroups.com>
> > .

[General Oya]

Derek and Russel,

TY for the tips re purification and restrictions. I was wondering if I would have to use alternative cut sites in order to produce the enzymes or not. I'll look into the yeast for certain.

Our PCR's got delayed a week so I'm still waiting to do the point mutation this week. I'll give you an update in the meantime, and we'd be happy to trade up and test each other if that might be helpful as well. Team Baltimore-US.

Gotta run to a Bio on the Bay networking event here in the harbor.

Take Care,
Ryan

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[Tom Randall]

> Have really highly competent cells and possibly try several Taq
> suppliers: "transform" cells with the protein prep. Virtually every
> recombinant protein prep is contaminated at a very low level with
> the recombinant plasmid. Since it is not known what resistance
> marker was used, plate on amp, kan and cam plates.
>
> I have used this trick to get several genes.
>
> DK

One might also be able to screen various Taq preps by PCR to select an
appropriately plasmid contaminated batch by using either M13 F and R
primers (most plasmids should have this) or more specifically, primers
specific to the Taq ORF itself. This should be a pretty sensitive
screen. I have tried several Taq suppliers and have never seen a no
DNA control with the standard M13 primers result in a band, so Taq
specific primers would probably be the way to go. Could save money on
expensive competent cells.

[Tito Jankowski]

What are other specific products besides Taq that this would work for?

Tito

[Cathal Garvey]

It might work for restriction enzymes, but only if:
1) The methylase is on the same plasmid or similarly contaminates the sample
2) The methylase gets to work before the restriction enzyme can, preserving the native genome from chopping.

Someone suggested either here or on the methods list that yeast expression of restriction enzymes with an export signal would save some of the trouble with methylases, a suggestion I consider rather excellent. It would also make purification of the protein far simpler. The only concern is whether the protein would survive in the extracellular atmosphere without a good buffer, or whether roaming proteases would kill it. Modifications to the core sequence such as addition of cysteines might help prevent denaturation, but you're getting into gene synthesis, defeating the purpose.

Bottom line, restriction enzymes might not work if the methylases don't act quickly enough, as they'd be lethal to an uprotected cell.

Ligase, phosphatase, and perhaps even insulin might work however. Insulin would make an interesting example for DIYbio if so, but you'd have to seriously caution against homebrew for medical use.

On 30 July 2010 15:56, Tito Jankowski <titoja...@gmail.com> wrote:

What are other specific products besides Taq that this would work for?

Tito

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[sgt york]

> Someone suggested either here or on the methods list that yeast expression
> of restriction enzymes with an export signal would save some of the trouble
> with methylases, a suggestion I consider rather excellent. It would also
> make purification of the protein far simpler. The only concern is whether
> the protein would survive in the extracellular atmosphere without a good
> buffer, or whether roaming proteases would kill it. Modifications to the
> core sequence such as addition of cysteines might help prevent denaturation,
> but you're getting into gene synthesis, defeating the purpose.

Just thinking randomly on this....

You wouldn't need an export tag. The yeast genome would be protected
from the RE by the nuclear envelope. Just have the stuff overexpressed
and harvest it from inside the yeast.