Disarming Agrobacterium

[mostromundo]

I'm looking into using Agrobacterium to transform a plant tissue
culture, can anyone suggest a place to look for procedures to disarm
Agrobacterium, or possibly a place to buy it already disarmed? I've
looked around some and so far haven't had much luck.

Thanks,

Micah

[Cory Tobin]

What do you mean by disarming it? Remove the virulence plasmid? If
you removed the virulence plasmid it would no longer be able to
transform the plant.

-Cory

[Frankco]

Yes you have to disarm it so it does not cause cancerous growth in the
plant.

Frank N.W. Indiana

[Frankco]

Since Agrobacterium cannot be transported across state lines in the U.S. I
think it would be possible to isolate a strain from the gall growth of
plants which you can find by just taking a walk in the woods. I've seen it
many times before but, before I was interested in obtaining it.

Google searches for "Agrobacterium tumefaciens disarm" brings up many
protocols. There is a patent on modifying Agrobacterium and it reveals some
information on the methods of disarming the Ti plamid.
http://www.freepatentsonline.com/EP0174166.html

There is so much potential in genetically modified plants, as scary as it
seems to some people - biofuels, land mine detectors and something as simple
as aesthetic value, the possibilities are endless.

Another vector for gene transfer might be pollen. Genetically modifying
prior to fertilization might be a good method of genetically modifying
plants.

Frank N.W. Indiana


----- Original Message -----
From: "Cory Tobin" <cory....@gmail.com>
To: <diy...@googlegroups.com>
Sent: Thursday, March 12, 2009 4:26 PM
Subject: Re: Disarming Agrobacterium


>

[Nathan McCorkle]

I bet agrobacterium gets transferred over state lines all the time and people never even know what they're doing... think soil, firewood, eh, who knows.

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

[Rajagopal]

Can any b ody suggest some plastic eating bacterium? how to collect and grow the bacterium?

[Nathan McCorkle]

http://www.ncbi.nlm.nih.gov/pubmed/18164311

[Aaron Hicks]

I think this kid just inoculated garbage with garbage juice from waste sites, and weighed the material (shredded plastic bags, I think) over time to see by how much the mass diminished.

http://theoystersgarter.com/2008/05/25/plastic-munching-bacteria-isolated-by-high-school-student/

Google up his name for some better descriptions of his work.

-AJ

[Nathan McCorkle]

i just heard about this from a totally different source yesterday

[Jake]

Do you have a reference for not being able to transport
agrobacterium? If it's just the vague "can't transport crop diseases"
laws, well those either don't apply, they aren't enforced, or they are
simply ignored.

Anyhow, I've got access to disarmed agro. If there's no major problem
that I'm not aware of with respect to shipping it out... I'd be happy
to donate some to the cause on the condition that recipients further
share it with the rest of the group for reasonable distribution costs.

-Jake

On Mar 12, 5:50 pm, "Frankco" <f...@frankandjackie.com> wrote:
> Since Agrobacterium cannot be transported across state lines in the U.S. I
> think it would be possible to isolate a strain from the gall growth of
> plants which you can find by just taking a walk in the woods. I've seen it
> many times before but, before I was interested in obtaining it.
>
> Google searches for "Agrobacterium tumefaciens disarm" brings up many
> protocols. There is a patent on modifying Agrobacterium and it reveals some

> information on the methods of disarming the Ti plamid.http://www.freepatentsonline.com/EP0174166.html

>
> There is so much potential in genetically modified plants, as scary as it
> seems to some people - biofuels, land mine detectors and something as simple
> as aesthetic value, the possibilities are endless.
>
> Another vector for gene transfer might be pollen. Genetically modifying
> prior to fertilization might be a good method of genetically modifying
> plants.
>
> Frank N.W. Indiana
>
>
>
> ----- Original Message -----
> From: "Cory Tobin" <cory.to...@gmail.com>
> To: <diy...@googlegroups.com>
> Sent: Thursday, March 12, 2009 4:26 PM
> Subject: Re: Disarming Agrobacterium
>
> >> I'm looking into using Agrobacterium to transform a plant tissue
> >> culture, can anyone suggest a place to look for procedures to disarm
> >> Agrobacterium, or possibly a place to buy it already disarmed? I've
> >> looked around some and so far haven't had much luck.
>
> > What do you mean by disarming it?  Remove the virulence plasmid?  If
> > you removed the virulence plasmid it would no longer be able to
> > transform the plant.
>

> > -Cory- Hide quoted text -
>
> - Show quoted text -

[mostromundo]

That's awesome, can I ask where you have access from?

On Mar 20, 2:34 pm, Jake <jakes...@mail.com> wrote:
> Do you have a reference for not being able to transportagrobacterium?  If it's just the vague "can't transport crop diseases"

> laws, well those either don't apply, they aren't enforced, or they are
> simply ignored.
>
> Anyhow, I've got access to disarmed agro.  If there's no major problem
> that I'm not aware of with respect to shipping it out...  I'd be happy
> to donate some to the cause on the condition that recipients further
> share it with the rest of the group for reasonable distribution costs.
>
> -Jake
>
> On Mar 12, 5:50 pm, "Frankco" <f...@frankandjackie.com> wrote:
>
>
>

> > SinceAgrobacteriumcannot be transported across state lines in the U.S. I

> > think it would be possible to isolate a strain from the gall growth of
> > plants which you can find by just taking a walk in the woods. I've seen it
> > many times before but, before I was interested in obtaining it.
>

> > Google searches for "Agrobacteriumtumefaciens disarm" brings up many
> > protocols. There is a patent on modifyingAgrobacteriumand it reveals some

> > information on the methods of disarming the Ti plamid.http://www.freepatentsonline.com/EP0174166.html
>
> > There is so much potential in genetically modified plants, as scary as it
> > seems to some people - biofuels, land mine detectors and something as simple
> > as aesthetic value, the possibilities are endless.
>
> > Another vector for gene transfer might be pollen. Genetically modifying
> > prior to fertilization might be a good method of genetically modifying
> > plants.
>
> > Frank N.W. Indiana
>
> > ----- Original Message -----
> > From: "Cory Tobin" <cory.to...@gmail.com>
> > To: <diy...@googlegroups.com>
> > Sent: Thursday, March 12, 2009 4:26 PM
> > Subject: Re: DisarmingAgrobacterium
>

> > >> I'm looking into usingAgrobacteriumto transform a plant tissue

> > >> culture, can anyone suggest a place to look for procedures to disarm
> > >>Agrobacterium, or possibly a place to buy it already disarmed? I've
> > >> looked around some and so far haven't had much luck.
>
> > > What do you mean by disarming it?  Remove the virulence plasmid?  If
> > > you removed the virulence plasmid it would no longer be able to
> > > transform the plant.
>
> > > -Cory- Hide quoted text -
>

> > - Show quoted text -- Hide quoted text -

[Jake]

On Mar 24, 9:46 pm, mostromundo <mzuor...@gmail.com> wrote:
> That's awesome, can I ask where you have access from?

A colleague of mine works with it in his lab.

Now I've been thinking a bit about the best way to go about this. I
need to find out what the laws and regulations on the matter are.
Normally I think labs just do what they want. I think there is some
rule about having a permit from the USDA for transporting crop
pathogens. Now I'm not sure if disarmed agro fits that category since
I don't think it could survive in the wild and being engineered it's
not really the same as the wild type "crop disease". I don't even
know if WT agro is considered a crop disease since I don't think it
kills any plants or really even harms them to any extent.

However, being a public forum, it's possible that some republicrat
fearmonger dillhole will see this and raise holy hell that we're
sending around and organism that injects foreign DNA into plants. If
that happens they could probably get government agencies to
investigate and charge us with some bullshit violation on the books
somewhere that isn't normally enforced and nobody else worries about.

So before going any further I'd like to find out what the laws are,
what the risk is, and how we'll deal with mitigating the legal and
biological risk. As a community we need to have some rules in place
so that it doesn't look like we're just sending things around without
any safeguards.

I think one of the first rules should be that people need to follow
standard lab protocols. Specificaly that organisims aren't released
into the wild. We need to be sure that people have the facilities to
deal with the organisims they work with and that they're sterilizing
all of their lab waste rather than throwing it out with the trash or
running it down a garbage disposal.

The last thing we need is a scare about "mutant monsters from the
dump" or "mutated swamp creatures from the sewers".


-Jake

[Aaron Hicks]

I think you're looking for 7 CFR 330. Happy hunting.

http://frwebgate3.access.gpo.gov/cgi-bin/TEXTgate.cgi?WAISdocID=0078463897+15+1+0&WAISaction=retrieve

Dialing back the language a touch and not being cavalier about interstate transport of plant pathogens might be in order.

Just a humble suggestion. My days of dabbling with agro are well behind me now.

-AJ

[mostromundo]

Hmm, according to CFR Title 7, 340.4 you need to apply for a Limited
Permit for Interstate Movement to transport genetically modified
Agrobacterium across state lines (I believe it states that any member
of any of the listed genera that has been genetically modified
applies, but I'm no lawyer so it's hard to say), it looks like a
pretty gigantic hassle. But, I didn't find anything about intrastate
transfers. Guhh, who writes these cfr's?

[Daniel C.]

On Thu, Mar 26, 2009 at 3:28 PM, mostromundo <mzuo...@gmail.com> wrote:
> But, I didn't find anything about intrastate transfers. Guhh, who writes these cfr's?

That actually makes sense. Since it deals with interstate transfers,
it's presumably a federal law. Each individual state would have to
make its own laws regarding transporting stuff within its own borders.

A cheat sheet saying what you can and can't do in each state would be
great. Who wants to make one? (I'm guessing "nobody" because it's
going to be such a pain.)

-Dan

[Jake]

I don't think most states have much in the way of regulation. I'm
pretty sure they're leaving it up to the feds. It really wouldn't
matter that much what a state legislated if the bordering states
didn't have the same regulation and it would only serve to discourage
scientific research in that state.

As far as the CFR mentioned above... It doesn't seem to specify if
agrobacterium is a "regulated article".

More info...

In Sec. 403 of the Plant Protection Act (7 U.S.C. 7701 et seq.),
``plant pest'' is defined as any living stage of any of the following
that can directly or indirectly injure, cause damage to, or cause
disease in any plant or plant product: A protozoan, a nonhuman
animal,
a parasitic plant, a bacterium, a fungus, a virus or viroid, an
infectious agent or other pathogen, or any article similar to or
allied
with any of the foregoing.


That's pretty darn vague. "Similar to" is entirely arbitrary and
relative. I'd guess that they would probably consider agro in this
category.

But at least we know what we need to do here now. It doesn't sound
all that hard to get a permit, just a lot of time consuming bullshit.


-Jake

[Frankco]

Hi Jake,

Yes the Fed or rather the Animal and Plant Health Inspection Service,
(APHIS), control the movement of certain genetically engineered organisms
including agrobactium, genetically modified plants and animals. I think the
APHIS is more interested in some genetically engineered organism being
released in to the wild than what you are working on in your lab. They want
to strictly control this to prevent any disasters from happening.

This is for genetically engineer organisms leaving your lab and being
introduced in to the wild:
"All regulated introductions of GE organisms must be authorized by APHIS
under either its permitting or notification procedures"
"APHIS uses the term biotechnology to mean the use of recombinant DNA
technology, or genetic engineering (GE) to modify living organisms."

This deals more with live stock.
APHIS' Veterinary Services', National Center for Import Export regulates the
import, export, and interstate movement of all animals and animal products
(e.g., tissues, blood, and semen), including those that are genetically
engineered.

I guess you will find this out after submitting to get a permit. They are
not to clear on what other agencies may be involved for different
genetically engineered organisms.
"In addition to permits for genetically engineered organisms, there may be
other agency requirements based upon other characteristics of the organism
or product."

I would think that someone in the group would be very familiar with these
procedures working in labs and having to obtain permits. No one?

You can submit for a permit on-line here. Again, I hear it's next to
impossible for someone not associated with an institution to get one. That
may be just hearsay though and may just be for the introduction of a
genetically engineered organism outside of the lab. Not sure.
http://www.aphis.usda.gov/permits/

Frank N.W. Indiana


----- Original Message -----

From: "Jake" <jake...@mail.com>
To: "DIYbio" <diy...@googlegroups.com>
Sent: Thursday, March 26, 2009 7:01 PM
Subject: Re: Disarming Agrobacterium


>

[Cory Tobin]

> I would think that someone in the group would be very familiar with these
> procedures working in labs and having to obtain permits. No one?

Most labs (or at least all the labs I'm familiar with) working with
agro never get permits because they never actually send bacteria.
Usually they just send purified plasmids in the mail. Upon receiving
the plasmid you just transform the bacteria yourself. This saves the
hassle of dealing with a government agency. In this case the only
paperwork is the institution's MTA.

-Cory

[Jeswin John]

I don't really understand this talk of organism transoprt. Is it really necessary. My belief is that instead of doing this, work on making the knowledge of the procedures available so others interested can do it in their lab.

Comparing with computers: When I first heard of OS hardware, I thought, "Hey, maybe I can get actual products" but rather it is the schematics and procedures that are available. Apply that to biotechnology, and we're fine.

[Frankco]

Thank you John.

 

Your post is very helpful. You're right, perhaps too much concern about shipping genetically engineered organisms.

 

Frank N.W. Indiana

 

 

----- Original Message -----

From: Jeswin John

To: diy...@googlegroups.com

Sent: Friday, March 27, 2009 7:03 AM

Subject: Re: Disarming Agrobacterium

[mostromundo]

> I don't really understand this talk of organism transoprt. Is it really
> necessary. My belief is that instead of doing this, work on making the
> knowledge of the procedures available so others interested can do it in
> their lab.

That's a good point, since the whole idea is do-it-yourself, but
there's something to be said for not reinventing the wheel. If
disarming Agro is really complicated it would be useful to have a
supply of it already disarmed for the DIYbio community to have access
to.

[Naomi Hernandez]

My first thought was that most labs just get the plasmids and do the transformations themselves; that's what's happened in every lab I've been in.  Except that that isn't actually true at least for the vast, vast majority of labs.  Yes, everyone gets their plasmids for their gene of interest separately and transforms but most of us start off with BL21 or some such E. coli strain which are purchased and shipped.  While they have no gene of interest per se they are tremendously genetically modified in order to create a receptive host organism with nice features.  Many have some sort of innate antibiotic resistance, some have rare codons, etc.

 

Things like antibiotic resistance and the like can be engineered into WT bacteria.  That's what all these corporations started out with if you go back far enough.  Most labs don't because it takes a lot of time and money to essentially re-invent the wheel.  (Although my guess would be that the genomes and associated plasmids to these strains are available somewhere.)

 

I know the question involved isn't about having stocks of E. coli that can then be transformed, it's about a specific strain doing specific things, but agrobacteria are fundamental to a particular kind of science the way E. coli are fundamental to another.  It seems that establishing fundamentals to be able to proceed in the experimentation that's actually the heart of the project should therefore be important to diybio in helping other people - and ourselves, for that matter - to have optimized protocols so that we can proceed to the work we really want to do.  And establishing these things ourselves so we don't have to purchase them ready made and - probably - overpriced seems to be in keeping with the whole diybio philosophy.

 

Anyway, that was a very long-winded way of saying that I agree with Cory.  Although the question remains, how do we get ungenetically modified cell strains and what counts as genetically modified?

[Jake]

Good points. We shouldn't have to reinvent the wheel every time we
want to do something. Whithout access to the tools of the trade it's
going to be a long road.

Maybe some of the chapters with facilities and institutional ties
should start working on getting permits and stocking some of these
strains. Everything has to be done on the up and up. Anyone who's
against GE is going to see us as the weak link in the chain and do
their best to shut us down. So we really need to be careful that some
minor rule breaking somewhere doesn't trip us up.


-Jake

[Naomi Hernandez]

Actually what I was thinking was if people wanted to re-invent the wheel this once and then have those protocols - and stocks - at our disposal for people in other diy situations or down the road.  Would anyone be interested in doing that aside from me?

[Aaron Hicks]

Note also that just having the transgenic agro is the tip of the iceberg. From that point, you have plants that have been transformed with the organism, and- despite rigorous attempts at disinfestation after that point- there is no guarantee that the resulting plants are free of transgenic agro.

As a result, you then need appropriate biosafety measures to ensure there is no transfer of material from the work into the wild- that means no potentially agro-contaminated material to sink or drain or trash, so you have to autoclave all of your waste. Once you have plants, they need to be grown in appropriate environments, once again with no waste (dead leaves, drain water, used medium) to sink, drain, or trash without autoclaving.

Then what do you do with the plants? Can't put them outside, and if they're anything other than ephemeral, determinate plants, you have to have a containment greenhouse for them.

Or, at least, that's how we had to do it, based on the regulations under which we operated in the United States. Somehow I doubt the USDA would take a pass on people who did this at home simply because they didn't have the wherewithall to do it properly.

-AJ

[Daniel C.]

On Fri, Mar 27, 2009 at 12:06 PM, Aaron Hicks <aaron...@gmail.com> wrote:
> Once you have plants, they need to be grown in appropriate environments,
> once again with no waste (dead leaves, drain water, used medium) to sink,
> drain, or trash without autoclaving.

So plants that you've used agrobacterium on can, separate from the
agro itself, transform other plants? Or does the agrobacterium take
up residence in the plant and become systemic?

-Dan

[Cory Tobin]

> So plants that you've used agrobacterium on can, separate from the
> agro itself, transform other plants?  Or does the agrobacterium take
> up residence in the plant and become systemic?

The first generation transformed plants have residual agro on them but
I've never heard of the residual bacteria on one plant transforming
another plant. The second generation is clean though. I've tried to
start a culture from 2nd gen plants before and it doesn't work. 1st
generation works just fine. Although, even though there is no more
agro after the second generation you still have to autoclave the
plants before throwing them out since they are transgenic.

-Cory

[Aaron Hicks]

We were required to maintain subsequent generations in secure greenhouses. I don't know if that was regulatory or overkill, but given that it would have been much cheaper to grow them conventionally, I suspect it was regulatory. It wasn't because the plants were exceptionally financially valuable as the case might be in industry.

It is unlikely, and generally considered a theoretical risk, that there may be some rogue organisms that persist past the T0 (transformation zero) generation plants even after rigorous antibiotic selection. Whether or not they persist on the T1 (first generation of progeny from the T0 plants) is well above my pay grade. Whenever queried about it, the illuminati would hem and haw and then go "Oooh! Donuts!" and scuttle away. It wasn't a question they liked to consider. I do recall one of them muttering something about studies showing presence of the DNA via PCR, but the details elude me.

But, yes- all transgenic material must be autoclaved. This serves several purposes:

1) Deactivation of any existing agro
2) Assured death of any propagatable material
3) Destruction of any remaining antibiotics
4) Denaturing most (all?) of the proteins produced in transgenic plants

I seem to recall we used 45 minute cycles at 15 psig before the material could be treated as conventional waste, i.e.: to landfill. Sharps (pipette tips and disposable pipettes) were boxed and sent for incineration.

I mean, yeah- I'm all in favor of do-it-yourself stuff, but homebrew agro work is made difficult by the level of containment required to do it properly.

-AJ

[Frankco]

Thanks Aaron for shedding light on the procedures and restrictions about dealing with agro.

 

Since I'm probably not going to do research to develop a plant that will benefit all humankind, I think, for me at least, agro is out of the question. I'll leave that up to qualified institutions or individuals who can work it and stay within the proper guidelines.

 

Do you know if there are any regulations on protoplast fusion, specifically interspecific protoplast fusion? In a sense the end results are genetically modified plants.

 

Frank N.W. Indiana

 

 

----- Original Message -----

From: Aaron Hicks

To: diy...@googlegroups.com

Sent: Friday, March 27, 2009 3:24 PM

Subject: Re: Disarming Agrobacterium

[Aaron Hicks]

Phew. None that I know of. The problems with GMO'd plants lie in the vector- plant pathogens such as A. tumefaciens and A. rhizogenes. Mashing together bits and pieces of otherwise unregulated species isn't a concern, provided there is no problematic vector involved.

There are other vectors for plant transformation; I know TbMV (tobacco mosaic virus) has been used. Supposedly it is "disarmed," but given the number of virus particles involved and how nothing is 100%, I maintain both my ignorance and skepticism as to its safety.

-AJ

[Frankco]

Thanks Aaron.

 

Pollen looks like a good candidate for agro replacement. I think it would be relatively safer and easier than using agro or TbMV. It's easy to get and pollen should be able to do just about anything that agro can do, just a different procedure.

 

The nice thing about pollen is that after germinating it lacks a small portion of the cell wall near its tip. No need for expensive enzymes to dissolve the cell wall or figuring out which combinations/concentrations of enzymes to use without destroying the cells. Introduction of exogenous DNA can be done with electroporation. I looks like it is a simpler method worthy of doing some tests.

 

A number of plants have been transformed this way. Here a protocol on tobacco transformation using pollen.

http://kasetsartjournal.ku.ac.th/kuj_files/2008/A0805111138117805.pdf

[Nathan McCorkle]

Definitely looks cool, it looks like the paper is from '94, I'm looking now for newer work. I wonder if the electroporators of today are any better with efficiencies? (listed here as 3.8%)

I wonder how it slips into the genome?

[Jake]

pCambia is doing a lot of work with agro replacements. They are
working on other agro-like bacteria in order to get around all the
patent issues with agro (it's all been patented like crazy). They
also stock vectors and the like.

http://www.cambia.org/

Last time I checked they were also looking for local distributor labs
to help disseminate their plasmids/vectors/etc.. That might be a good
way to get access to everything at once and also help out their
project.


As far as what Aaron Hicks was saying... It was explained to me at
some point that there is a lot of holdover from when they first
started working with agro and didn't know enough about it to determine
the safety. They were also once working with non-disarmed strains.

But now that agro has been worked with extensively I think most labs
are a lot more lax on procedures. You still have to sterilize
everything. You can't allow even a single pollen grain out of your
lab or you chance your transgenic DNA escaping into the wild. The
concerns about the agro escaping aren't really serious. Disarmed agro
can't survive in the wild. It has no T-DNA which would allow it to
infect or form a symbiosis with wild plants. It can only survive in
the lab where it is fed nutrients. Even if some did get in the soil
and survive for a bit or in low numbers it still wouldn't be able to
reach significant numbers without being able to use a plant host. In
the worst case senario if a stray agro did manage to infect a plant
cell it still wouldn't be getting any return from it's interaction and
the result would simply be a single transformed plant cell somewhere
in the plant's root. The transgenic root cell would just live out
it's life and eventually die.

The chances of your agro getting out and doing anything at all rely on
a dozen million to one events happening at the right times and even
then nobody would ever know and there would be no consequences. OTOH
if you don't follow proper procedures with your transgenic plants you
could EASILY be releasing transgenic DNA into the environment. If a
single pollen grain gets out of your lab or secure greenhouse it could
fertilize the wild-type of your organism and cause problems. Even if
the pollen grain lands on the ground or on a different species bees
and other insects could pick it up and transport it to a compatible
species.

All it would take to get shutdown forever and the science regulated to
death would be for a few GFP plants to turn up around a DIYbio lab
somewhere.

That's why it doesn't really matter how you transform something. Agro
is just the easiest and cheapest way to go about it. We better just
get used to sterilizing everything and doing it small-scale enough to
make sure everything is contained. In that respect agro works well.
We can't use methods that require screening hundreds or thousands of
plants. There's just no way to keep all of those contained at a
reasonable cost.


As far as protoplast fusion... That is not considered genetic
engineering. It's considered the same as a hybrid and you don't have
to treat it like a GMO. If you develop something it also doesn't have
to be labeled as a GMO in countries that require it. Plenty of useful
strains have been developed that way, at least for fungi. I don't
know much about it's use in other kingdoms.


-Jake