Anyone working with plant protoplasts?
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Sebastian Cocioba
Jun 17, 2013, 6:13:50 PM6/17/13
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Its my research focus (private non-academic) and I'm working on a protoplast regeneration pipeline for PEG mediated transformation. I'm going to publish the protocols to openwetware.org once finished. Its in much need of more plant based protocols. This is a shout out to anyone interested or is currently working with plants. Wanna share notes? I'd love to collaborate on any work involving these sensitive little guys or plants in general.
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Sung won Lim
Jun 17, 2013, 6:15:22 PM6/17/13
to Diybio
Seconded. Would love to compare notes
-sung
On Jun 17, 2013 6:14 PM, "Sebastian Cocioba" <scoc...@gmail.com> wrote:
Its my research focus (private non-academic) and I'm working on a protoplast regeneration pipeline for PEG mediated transformation. I'm going to publish the protocols to openwetware.org once finished. Its in much need of more plant based protocols. This is a shout out to anyone interested or is currently working with plants. Wanna share notes? I'd love to collaborate on any work involving these sensitive little guys or plants in general.
Sent from my Windows Phone
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Cory Tobin
Jun 18, 2013, 5:06:38 PM6/18/13
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I'd be happy to share notes on general plant stuff, although I haven't
made protoplasts in ~5 years and I could never maintain protoplasts in
suspension for very long. The only thing I could ever get to work
reliably was to create the protoplasts then very rapidly isolate the
nuclei, although this is irrelevant for transformation/regeneration.
Why do you want to do PEG transformation? I haven't heard of anyone
doing this in like 10 years. Agro infiltration and particle
bombardment are usually the preferred methods.
-cory
made protoplasts in ~5 years and I could never maintain protoplasts in
suspension for very long. The only thing I could ever get to work
reliably was to create the protoplasts then very rapidly isolate the
nuclei, although this is irrelevant for transformation/regeneration.
Why do you want to do PEG transformation? I haven't heard of anyone
doing this in like 10 years. Agro infiltration and particle
bombardment are usually the preferred methods.
-cory
Nathan McCorkle
Jun 18, 2013, 5:26:39 PM6/18/13
to diybio
On Tue, Jun 18, 2013 at 2:06 PM, Cory Tobin <cory....@gmail.com> wrote:
> Why do you want to do PEG transformation?
It's highly available???
> Why do you want to do PEG transformation?
somewhat unrealted:
When I made hybridomas I believe we used PEG, so the techniques for
protoplast handling would likely directly translate since the cells
are highly susceptible to mishandling, like protoplasts.
-Nathan
Cathal Garvey (Android)
Jun 18, 2013, 6:07:38 PM6/18/13
to diy...@googlegroups.com
Agro often controlled, bombardment really expensive? A reliable-ish PEG method would definitely suit me better. :)
--
Sent from my Android phone with K-9 Mail. Please excuse my brevity.
Sent from my Android phone with K-9 Mail. Please excuse my brevity.
Cory Tobin
Jun 18, 2013, 6:37:14 PM6/18/13
to diybio
I see. Makes sense now.
Yeah, now that I think about it both of those methods would be
difficult to DIY. PEG transformation would be useful for DIY
biologists.
-cory
Yeah, now that I think about it both of those methods would be
difficult to DIY. PEG transformation would be useful for DIY
biologists.
-cory
John Griessen
Jun 18, 2013, 7:05:33 PM6/18/13
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John Griessen
Jun 18, 2013, 7:10:47 PM6/18/13
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On 06/18/2013 04:06 PM, Cory Tobin wrote:
> Why do you want to do PEG transformation? I haven't heard of anyone
> doing this in like 10 years. Agro infiltration and particle
> bombardment are usually the preferred methods.
A quick googling turned up this free published article with an intro section on why:
> Why do you want to do PEG transformation? I haven't heard of anyone
> doing this in like 10 years. Agro infiltration and particle
> bombardment are usually the preferred methods.
http://mpipz.iwww.mpg.de/25781/Mathur_PEG_transformation.pdf
Not that I'm experienced in this... but...
Nathan McCorkle
Jun 18, 2013, 7:33:38 PM6/18/13
to diybio
Well if you're talking building equipment, it would more work upfront,
but probably be higher efficiency for transformation and likely a lot
easier to shoot DNA ballistically.
--
-Nathan
but probably be higher efficiency for transformation and likely a lot
easier to shoot DNA ballistically.
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-Nathan
Nathan McCorkle
Jun 18, 2013, 11:07:41 PM6/18/13
to John Griessen, Sebastian Cocioba, diybio
On Tue, Jun 18, 2013 at 5:44 PM, John Griessen <jo...@industromatic.com> wrote:
> after a blast?
Not sure exactly, Patrik has posted some jove tutorials/videos, and
someone made a DIY gene gun and got onion cells to glow:
http://diyhpl.us/~bryan/papers2/diybio/r%C3%BCdiger-trojok-gene-gun.pdf
>
> Can one separate the spatter of the blast from the gun b closing a door?
Probably, it's an air gun firing 1 micron gold particles coated with
DNA (add wet DNA solution, then dry, then shoot)
>
> Sounds like lots of engineering.
Maybe not, Sebastian posted this to me (maybe accidentally, you also
only mailed me just now) which sounds MUCH more involved and finicky.
There are pros and cons to both routes I'm sure, maybe one method is
better for the plant genome where the other targets the chloroplast
(have you heard that chloroplasts and mitochondria are the result of
endosymbiosis long long ago... i.e. a small cell living in a big
cell).
>
> PEG sounds like another process add-on to my
> incubator/mild-centrifuge/liquid-handling/air-PCR-performing/OD-measuring
> 15cm x 20cm x 10cm robot idea.
Maybe, see below. I would think that with enough energy, you could
make both devices and have two separate and complementary products.
On Tue, Jun 18, 2013 at 5:53 PM, Sebastian Cocioba <scoc...@gmail.com> wrote:
> The mathur article is a great method for transformation but the steps
> to achieve happy protoplasts is much more difficult. The goal is to
> obtain clean, healthy cells and the best way so far is by ficoll or
> sucrose gradient centrifugation. The catch is the need for a swing out
> centrifuge so the protoplast separate into clear bands at the interface
> of a two or three step gradient. Its quite difficult to alter the
> density of a solution while maintaining isoosmotic conditions. Ficoll
> is a great solution but its way too expensive per rxn. Any sucrose
> polymer that won't be taken up or is metabolically inert would work.
How about any of the sugar alcohols, or maybe short-branches
polysaccharides, gums?
> Sung won Lim and I have been brainstorming on this topic for a while
> now and are in the process of testing different methods. Just keeping
> these guys alive is proving to be the fundamental problem. I can see
> why people have all but abandoned the technique. Devising a good
> protocol would be a great boon for DIY plant bio so I see it as a
> worthwhile endeavor. Nathan, could you elaborate on your work? How was
> the handling of that particular organism similar? Thanks.
Well for hybridoma production we took mouse spleen and rinsed it of
blood, ground it up and strained out the gristle/matrix, counted the
ratio of B cells to macrophages, then added PEG and the appropriate
amount of cancerous B-cells that were grown in culture. Slowly the
solution was mixed, and the PEG encouraged cell fusion, successful
hybridomas can be selected for using a gene defect in the cancerous
cell line, and the fact that non-cancerous B cells will naturally die
pretty fast in cell culture.
http://en.wikipedia.org/wiki/Hybridoma_technology
I mention it being similar because during PEG treatment, the cells are
quite unstable, so if you jostle them too much or stir the solution
too vigorously you simply break the cells open. I've had the same
experience with spheroplast preparation for transformations in
bacteria (either e.coli or b.subtilis, can't remember), mix too
hard/fast and you kill your little buddies.
--
-Nathan
> On 06/18/2013 06:33 PM, Nathan McCorkle wrote:
>>
>> Well if you're talking building equipment, it would more work upfront,
>> but probably be higher efficiency for transformation and likely a lot
>> easier to shoot DNA ballistically.
>
>
> Is it lower cost and reliable though? What are the cleanup steps needed
>>
>> Well if you're talking building equipment, it would more work upfront,
>> but probably be higher efficiency for transformation and likely a lot
>> easier to shoot DNA ballistically.
>
>
> after a blast?
Not sure exactly, Patrik has posted some jove tutorials/videos, and
someone made a DIY gene gun and got onion cells to glow:
http://diyhpl.us/~bryan/papers2/diybio/r%C3%BCdiger-trojok-gene-gun.pdf
>
> Can one separate the spatter of the blast from the gun b closing a door?
Probably, it's an air gun firing 1 micron gold particles coated with
DNA (add wet DNA solution, then dry, then shoot)
>
> Sounds like lots of engineering.
Maybe not, Sebastian posted this to me (maybe accidentally, you also
only mailed me just now) which sounds MUCH more involved and finicky.
There are pros and cons to both routes I'm sure, maybe one method is
better for the plant genome where the other targets the chloroplast
(have you heard that chloroplasts and mitochondria are the result of
endosymbiosis long long ago... i.e. a small cell living in a big
cell).
>
> PEG sounds like another process add-on to my
> incubator/mild-centrifuge/liquid-handling/air-PCR-performing/OD-measuring
> 15cm x 20cm x 10cm robot idea.
Maybe, see below. I would think that with enough energy, you could
make both devices and have two separate and complementary products.
On Tue, Jun 18, 2013 at 5:53 PM, Sebastian Cocioba <scoc...@gmail.com> wrote:
> The mathur article is a great method for transformation but the steps
> to achieve happy protoplasts is much more difficult. The goal is to
> obtain clean, healthy cells and the best way so far is by ficoll or
> sucrose gradient centrifugation. The catch is the need for a swing out
> centrifuge so the protoplast separate into clear bands at the interface
> of a two or three step gradient. Its quite difficult to alter the
> density of a solution while maintaining isoosmotic conditions. Ficoll
> is a great solution but its way too expensive per rxn. Any sucrose
> polymer that won't be taken up or is metabolically inert would work.
How about any of the sugar alcohols, or maybe short-branches
polysaccharides, gums?
> Sung won Lim and I have been brainstorming on this topic for a while
> now and are in the process of testing different methods. Just keeping
> these guys alive is proving to be the fundamental problem. I can see
> why people have all but abandoned the technique. Devising a good
> protocol would be a great boon for DIY plant bio so I see it as a
> worthwhile endeavor. Nathan, could you elaborate on your work? How was
> the handling of that particular organism similar? Thanks.
Well for hybridoma production we took mouse spleen and rinsed it of
blood, ground it up and strained out the gristle/matrix, counted the
ratio of B cells to macrophages, then added PEG and the appropriate
amount of cancerous B-cells that were grown in culture. Slowly the
solution was mixed, and the PEG encouraged cell fusion, successful
hybridomas can be selected for using a gene defect in the cancerous
cell line, and the fact that non-cancerous B cells will naturally die
pretty fast in cell culture.
http://en.wikipedia.org/wiki/Hybridoma_technology
I mention it being similar because during PEG treatment, the cells are
quite unstable, so if you jostle them too much or stir the solution
too vigorously you simply break the cells open. I've had the same
experience with spheroplast preparation for transformations in
bacteria (either e.coli or b.subtilis, can't remember), mix too
hard/fast and you kill your little buddies.
--
-Nathan
Sebastian Cocioba
Jun 18, 2013, 11:40:48 PM6/18/13
to diy...@googlegroups.com
Didn't hit reply all the first time. Silly windows phone. Anyway...
Mannitol, sorbitol et al (pun intended) work as well but do not feed
the cells. Its a delicate balance of inert to metabolically active
sugars that need replenishment as the food sugar (sucrose) depletes.
Did I mention it all needs to be sterile? The ficoll gradient spins
down everything except the protoplasts. One paper even cites
purposefully inoculating a protoplast culture with microbes and
comparing results. It gave a 95-100% removal of all contaminants
present. Its kinda magical. The Jove video was for P. patens which has
very little cellulose layers to eat through so it does not represent a
protocol for a broad range of tissue types. Different tissues need
different enzyme concentrations and types. I found macerozyme and
cellulase from Phytotech to work very well across genera. Sigma's
driselase is liquid gold but works beautifully. I'm waiting on an order
from a Chinese supplier of cellulase and hemicellulase. As a side
note...im not too fond of shooting gold down the drain. Maybe use FeCl2
to remove the gold coating off contacts of junk electronics and
electroplate Nickel dust with the gold micro flakes? I see aqua Regia
being needed which would make things much more complex but I digress.
Sent from my Windows Phone From: Nathan McCorkle
Sent: 6/18/2013 11:08 PM
To: John Griessen; Sebastian Cocioba; diybio
Subject: Re: [DIYbio] Anyone working with plant protoplasts?
Mannitol, sorbitol et al (pun intended) work as well but do not feed
the cells. Its a delicate balance of inert to metabolically active
sugars that need replenishment as the food sugar (sucrose) depletes.
Did I mention it all needs to be sterile? The ficoll gradient spins
down everything except the protoplasts. One paper even cites
purposefully inoculating a protoplast culture with microbes and
comparing results. It gave a 95-100% removal of all contaminants
present. Its kinda magical. The Jove video was for P. patens which has
very little cellulose layers to eat through so it does not represent a
protocol for a broad range of tissue types. Different tissues need
different enzyme concentrations and types. I found macerozyme and
cellulase from Phytotech to work very well across genera. Sigma's
driselase is liquid gold but works beautifully. I'm waiting on an order
from a Chinese supplier of cellulase and hemicellulase. As a side
note...im not too fond of shooting gold down the drain. Maybe use FeCl2
to remove the gold coating off contacts of junk electronics and
electroplate Nickel dust with the gold micro flakes? I see aqua Regia
being needed which would make things much more complex but I digress.
Sent from my Windows Phone From: Nathan McCorkle
Sent: 6/18/2013 11:08 PM
To: John Griessen; Sebastian Cocioba; diybio
Subject: Re: [DIYbio] Anyone working with plant protoplasts?
John Griessen
Jun 19, 2013, 2:38:33 PM6/19/13
to scoc...@gmail.com, diy...@googlegroups.com, Nathan McCorkle
On 06/18/2013 10:07 PM, Nathan McCorkle wrote:
> Probably, it's an air gun firing 1 micron gold particles coated with
> DNA (add wet DNA solution, then dry, then shoot)
>
>>
>> Sounds like lots of engineering.
>
> Maybe not
> Probably, it's an air gun firing 1 micron gold particles coated with
> DNA (add wet DNA solution, then dry, then shoot)
>
>>
>> Sounds like lots of engineering.
>
> Maybe not
>> PEG sounds like another process add-on to my
>> incubator/mild-centrifuge/liquid-handling/air-PCR-performing/OD-measuring
>> 15cm x 20cm x 10cm robot idea.
>
> Maybe, see below. I would think that with enough energy, you could
> make both devices and have two separate and complementary products.
Sure, and also, by design, the products have many common parts, keeping costs down.
>> incubator/mild-centrifuge/liquid-handling/air-PCR-performing/OD-measuring
>> 15cm x 20cm x 10cm robot idea.
>
> Maybe, see below. I would think that with enough energy, you could
> make both devices and have two separate and complementary products.
> On Tue, Jun 18, 2013 at 5:53 PM, Sebastian Cocioba wrote:
The goal is to
>> obtain clean, healthy cells and the best way so far is by ficoll or
>> sucrose gradient centrifugation. The catch is the need for a swing out
>> centrifuge so the protoplast separate into clear bands at the interface
>> of a two or three step gradient.
Are you saying the media to centrifuge is layers of different densities
carefully poured into a centrifuge vial?
Sebastian Cocioba wrote: Any sucrose
>> polymer that won't be taken up or is metabolically inert would work.
>> Just keeping
>> these guys alive is proving to be the fundamental problem.
>> these guys alive is proving to be the fundamental problem.
>> Devising a good
>> protocol would be a great boon for DIY plant bio so I see it as a
>> worthwhile endeavor.
>> protocol would be a great boon for DIY plant bio so I see it as a
>> worthwhile endeavor.
Nathan McCorkle wrote:
> I mention it being similar because during PEG treatment, the cells are
> quite unstable, so if you jostle them too much or stir the solution
> too vigorously you simply break the cells open. I've had the same
> experience with spheroplast preparation for transformations in
> bacteria (either e.coli or b.subtilis, can't remember), mix too
> hard/fast and you kill your little buddies.
Then maybe a mild slow centrifuge, plus some handler automation
> I mention it being similar because during PEG treatment, the cells are
> quite unstable, so if you jostle them too much or stir the solution
> too vigorously you simply break the cells open. I've had the same
> experience with spheroplast preparation for transformations in
> bacteria (either e.coli or b.subtilis, can't remember), mix too
> hard/fast and you kill your little buddies.
to prep the stepped gradients is a way to consider?
How could one use stepped gradients without a swing out centrifuge holder?
Use a gel so it could centrifuge sideways?
Swing out is higher danger, higher cost... harder to automate with cheap blind robots,
limits range of uses for a spindle. This kind of task might need a dedicated spindle
with swing out holders.
Still, swing out holders could be low cost if it ran at mild G's and took a while.
What time and G's separate protoplasts?
Cathal Garvey (Android)
Jun 19, 2013, 3:10:35 PM6/19/13
to diy...@googlegroups.com
Yes, layered gradient of different sucrose concentrations. Can be done in a fixed angle (say, 45 degrees) rotor with slow acceleration/deceleration but obviously not as well as with swinging buckets, and not at all in a horizontally fixed rotor like a dremelfuge.
Sebastian Cocioba
Jun 20, 2013, 2:30:45 AM6/20/13
to diy...@googlegroups.com
I'm gonna try the $70 hand cranked swing out centrifuge from amazon. The protoplasts need very little g force so maybe this could work. Maybe a hacked version with a nema stepper? Sure beats 500 bucks worth of equipment. The low speed should minimize any projectile damage to a mild bruise and maybe a broken window. Someone reviewed the crank and calculated a 260g force at 75rpm. It holds 4 15mL tubes. Seems perfect for the job. Ill chime in on Monday after a ficoll run using the crank. Hopefully this can overcome one of the many technical hurdles in the quest for perfect 'plasts.
Sent from my Windows Phone
Subject: Re: [DIYbio] Anyone working with plant protoplasts?
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Cathal Garvey (Android)
Jun 20, 2013, 2:39:21 AM6/20/13
to diy...@googlegroups.com
Link?
Sebastian Cocioba
Jun 20, 2013, 3:02:47 AM6/20/13
to diy...@googlegroups.com
http://www.amazon.com/dp/B006OCRE02/ref=cm_sw_em_r_am_wp_am_us?ie=UTF8
I think I snagged the last one. Carolina sells a similar device for ten dollars more.
I think I snagged the last one. Carolina sells a similar device for ten dollars more.
Sent from my Windows Phone
Sent: 6/20/2013 2:39 AM
To: diy...@googlegroups.com
Subject: RE: [DIYbio] Anyone working with plant protoplasts?
To: diy...@googlegroups.com
Subject: RE: [DIYbio] Anyone working with plant protoplasts?
-- You received this message because you are subscribed to the Google Groups DIYbio group. To post to this group, send email to diy...@googlegroups.com. To unsubscribe from this group, send email to diybio+un...@googlegroups.com. For more options, visit this group at https://groups.google.com/d/forum/diybio?hl=en
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Sebastian Cocioba
Jun 20, 2013, 3:08:18 AM6/20/13
to diy...@googlegroups.com
This one is fancy and in your neighborhood, cathal.
http://www.djblabcare.co.uk/djb/product/1136/Centrifuges-C1011-Hettich_Hand_Centrifuge
http://www.djblabcare.co.uk/djb/product/1136/Centrifuges-C1011-Hettich_Hand_Centrifuge
Sent from my Windows Phone
Cathal Garvey (Android)
Jun 20, 2013, 3:10:59 AM6/20/13
to diy...@googlegroups.com
Awesome, thanks!
Nathan McCorkle
Jun 20, 2013, 4:28:58 AM6/20/13
to diybio
Looks like you can order directly from that manufacturer/brand-name:
http://www.thomassci.com/Equipment/Centrifuges/_/Hand-Driven-Centrifuge/
--
-Nathan
http://www.thomassci.com/Equipment/Centrifuges/_/Hand-Driven-Centrifuge/
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