Chloroplast Transfection?
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Cathal Garvey
Dec 1, 2009, 5:04:47 AM12/1/09
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Hi all,
Has anyone here experience with or a protocol for the transfection of a Chloroplast in either plant or algal cells?
For that matter, has anyone worked with mitochondrial transfection, as the protocols are likely to be similar?
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Has anyone here experience with or a protocol for the transfection of a Chloroplast in either plant or algal cells?
For that matter, has anyone worked with mitochondrial transfection, as the protocols are likely to be similar?
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Cathal Garvey
Dec 1, 2009, 7:58:49 AM12/1/09
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I've since found that one can achieve chloroplast transfection using a gene gun.. but those things are obviously hard to get a hold of. Also, they'd still need a protocol.
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2009/12/1 Cathal Garvey <cathal...@gmail.com>
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Parijata Mackey
Dec 1, 2009, 9:57:24 AM12/1/09
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Hi there,
I'm not really sure what you're trying to do, nor your level of expertise, so please forgive the vague nature of this response. However, if you're googling for "chloroplast transfection" you're probably not going to find much; transfection is conventionally done to animal cells, transformation is what we do to plants.
The most common methods of chloroplast transformation are (1) the gene gun, (2) polyethylene glycol treatment, and (3) microinjection. The gene gun is by far the most popular; it's efficient, it's fast, it's versatile, and it's reliable (and just really cool). Unfortunately, it's also expensive (unless you have one lying around). Microinjection is exactly what you're picturing; you inject the DNA straight into the chloroplasts with a tiny syringe (0.1nm diameter). Cool stuff happens; galistan (a liquid metal) is heated and forces the plasmids through an unreasonably small capillary tip... but I suspect this equipment is also hard to get a hold of.
PEG was cool when it came out in the 1990s, but it's less efficient than shooting DNA-coated gold bullets at baby plants. However, it's by far the easiest and cheapest of the reliable methods; check it out. If I were trying to DIY-chloroplast-transform, that would probably be my protocol of choice. Of course, I'm not a plant biologist, so there could be some wild new method I don't know about. You could contact a researcher directly and ask them to make sure.
Other than that, mostly to satisfy the curiosity of us non-botanists, here are some general chloroplast protocols/book chapters: link, link, link, link.
And for even more generic information, here is:
Cheers,
Jata :-)
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Parijata Mackey
University of Chicago
pari...@uchicago.edu
www.parijata.com
“Seek freedom and become captive of your desires. Seek discipline and find your liberty.” --Frank Herbert
“I am so clever that sometimes I don't understand a single word of what I am saying.” --Oscar Wilde
I'm not really sure what you're trying to do, nor your level of expertise, so please forgive the vague nature of this response. However, if you're googling for "chloroplast transfection" you're probably not going to find much; transfection is conventionally done to animal cells, transformation is what we do to plants.
The most common methods of chloroplast transformation are (1) the gene gun, (2) polyethylene glycol treatment, and (3) microinjection. The gene gun is by far the most popular; it's efficient, it's fast, it's versatile, and it's reliable (and just really cool). Unfortunately, it's also expensive (unless you have one lying around). Microinjection is exactly what you're picturing; you inject the DNA straight into the chloroplasts with a tiny syringe (0.1nm diameter). Cool stuff happens; galistan (a liquid metal) is heated and forces the plasmids through an unreasonably small capillary tip... but I suspect this equipment is also hard to get a hold of.
PEG was cool when it came out in the 1990s, but it's less efficient than shooting DNA-coated gold bullets at baby plants. However, it's by far the easiest and cheapest of the reliable methods; check it out. If I were trying to DIY-chloroplast-transform, that would probably be my protocol of choice. Of course, I'm not a plant biologist, so there could be some wild new method I don't know about. You could contact a researcher directly and ask them to make sure.
Other than that, mostly to satisfy the curiosity of us non-botanists, here are some general chloroplast protocols/book chapters: link, link, link, link.
And for even more generic information, here is:
- A simple but decently cited powerpoint.
- A basic but decent overview of chloroplast engineering (strangely, written by a guy I used to know in high school... Shiv, are you on this list?).
- Some gene gun protocols (seriously, if you get your hands on one of those, tell us how you did it... and wear ear protection!).
Cheers,
Jata :-)
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Parijata Mackey
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“Seek freedom and become captive of your desires. Seek discipline and find your liberty.” --Frank Herbert
“I am so clever that sometimes I don't understand a single word of what I am saying.” --Oscar Wilde
Simon Quellen Field
Dec 1, 2009, 10:32:04 AM12/1/09
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Sounds to me like we should start a homebrew gene gun project...
My latest science fiction novel A Twisted Garden is now available in bookstores.
Simon Quellen Field
Dec 1, 2009, 10:58:24 AM12/1/09
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If you want to make a microinjector, I sell GalInStan on my web site:
"http://www.scitoyscatalog.com/Merchant2/merchant.mvc?Screen=PROD&Product_Code=LIQUIDMETAL&Category_Code=H"
You can make the micro-needle by pulling on a hot glass tube.
I don't believe it needs to be anything like 0.1nm -- that seems to be at least
three orders of magnitude smaller than necessary. A chloroplast is about
2 to 10 microns, so if you want to inject a whole chloroplast, you might want
to try a needle with a 10 micron inside diameter and perhaps a 20 micron
outside diameter. Plant cells can be 100 microns or even 400 microns
across, and you might want to start with the big ones anyway, for practice.
While GalInStan (an alloy of gallium, indium, and tin that is non-toxic but liquid)
is fun to play with, I would bet that a rubber bulb from an eye dropper would
give you enough control, especially if you pushed it all the way down onto the
glass so that there was a lot less motion available. You could then try sucking
chloroplasts out of one cell and sticking them into another.
It might be interesting to see what happens when you inject chloroplasts into
a plant like creeping dodder, which has lost chloroplasts as it evolved into a
parasite.
Another interesting project might be to inject C4 chloroplasts into C3 plants,
and see if they produce more sugar as a result. Sucking out all of the
chloroplasts might be difficult without sucking out all of the other internal
structures, but just injecting new ones might be enough. Or find a way to
kill the chloroplasts without killing the cell, and then inject new ones.
"http://www.scitoyscatalog.com/Merchant2/merchant.mvc?Screen=PROD&Product_Code=LIQUIDMETAL&Category_Code=H"
You can make the micro-needle by pulling on a hot glass tube.
I don't believe it needs to be anything like 0.1nm -- that seems to be at least
three orders of magnitude smaller than necessary. A chloroplast is about
2 to 10 microns, so if you want to inject a whole chloroplast, you might want
to try a needle with a 10 micron inside diameter and perhaps a 20 micron
outside diameter. Plant cells can be 100 microns or even 400 microns
across, and you might want to start with the big ones anyway, for practice.
While GalInStan (an alloy of gallium, indium, and tin that is non-toxic but liquid)
is fun to play with, I would bet that a rubber bulb from an eye dropper would
give you enough control, especially if you pushed it all the way down onto the
glass so that there was a lot less motion available. You could then try sucking
chloroplasts out of one cell and sticking them into another.
It might be interesting to see what happens when you inject chloroplasts into
a plant like creeping dodder, which has lost chloroplasts as it evolved into a
parasite.
Another interesting project might be to inject C4 chloroplasts into C3 plants,
and see if they produce more sugar as a result. Sucking out all of the
chloroplasts might be difficult without sucking out all of the other internal
structures, but just injecting new ones might be enough. Or find a way to
kill the chloroplasts without killing the cell, and then inject new ones.
Cathal Garvey
Dec 1, 2009, 12:50:33 PM12/1/09
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Hi Parijata,
Thanks so much for all that information! It's fantastic to have such a comprehensive set of references to explore.
I have access to very little resources but I have a pretty good education on plant transfection using either Agrobacterium or Gene-Gun approaches. My local University has a hacked-together Gene gun made of an air-compressed shotgun which I might convince them to let me use for my own work if I were to ask. They're pretty laid back.
As far as microinjecting, the difficulty of getting a needle small enough to pierce mitochondria/chloroplasts aside, I gather it's a very fine art. Apparently microinjecting ova when attempting IVF is difficult enough (and Ova are among the biggest cells naturally found in the body), so I don't fancy my chances with an organelle!
I can foresee a way of using Agrobacterium to transfect organelles, seeing as it simply uses NLS sequences to target the nucleus. Replacing these with a DNA sequence that an organelle's endogenous transcription factor targets would get it into the organelle provided the cell's own machinery made the transcription factor, and the factor's DNA targeting domain were active in the cytoplasm. However, I doubt this has been done yet. Plus, it wouldn't work for Algae anyway, as they aren't a legitimate target for Agrobacterium.
Regarding Transfection/Transformation, I was cagey about terminology as chloroplasts can be vaguely considered procaryotes/proto-bacteria themselves. :)
Many thanks for your help, and I'll let you know if anything comes of it at some stage down the line!
-Cathal Garvey
--
letters.cunningprojects.com
twitter.com/onetruecathal
Thanks so much for all that information! It's fantastic to have such a comprehensive set of references to explore.
I have access to very little resources but I have a pretty good education on plant transfection using either Agrobacterium or Gene-Gun approaches. My local University has a hacked-together Gene gun made of an air-compressed shotgun which I might convince them to let me use for my own work if I were to ask. They're pretty laid back.
As far as microinjecting, the difficulty of getting a needle small enough to pierce mitochondria/chloroplasts aside, I gather it's a very fine art. Apparently microinjecting ova when attempting IVF is difficult enough (and Ova are among the biggest cells naturally found in the body), so I don't fancy my chances with an organelle!
I can foresee a way of using Agrobacterium to transfect organelles, seeing as it simply uses NLS sequences to target the nucleus. Replacing these with a DNA sequence that an organelle's endogenous transcription factor targets would get it into the organelle provided the cell's own machinery made the transcription factor, and the factor's DNA targeting domain were active in the cytoplasm. However, I doubt this has been done yet. Plus, it wouldn't work for Algae anyway, as they aren't a legitimate target for Agrobacterium.
Regarding Transfection/Transformation, I was cagey about terminology as chloroplasts can be vaguely considered procaryotes/proto-bacteria themselves. :)
Many thanks for your help, and I'll let you know if anything comes of it at some stage down the line!
-Cathal Garvey
2009/12/1 Parijata Mackey <pari...@gmail.com>
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Aaron Hicks
Dec 1, 2009, 2:04:49 PM12/1/09
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On Tue, Dec 1, 2009 at 8:32 AM, Simon Quellen Field <sfi...@scitoys.com> wrote:
Sounds to me like we should start a homebrew gene gun project...
Not too tough, all in all. I seem to recall the early Dow (?) biolistics used a .22 blank, but I could be wrong about that. I've seen (never operated) the ones that run off a burst disk- very heavy aluminum and Plexi stuff.
-AJ
Nathan McCorkle
Dec 2, 2009, 5:54:59 AM12/2/09
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On Tue, Dec 1, 2009 at 10:58 AM, Simon Quellen Field <sfi...@scitoys.com> wrote:
If you want to make a microinjector, I sell GalInStan on my web site:
"http://www.scitoyscatalog.com/Merchant2/merchant.mvc?Screen=PROD&Product_Code=LIQUIDMETAL&Category_Code=H"
You can make the micro-needle by pulling on a hot glass tube.
I don't believe it needs to be anything like 0.1nm -- that seems to be at least
0.5 - 5 microns
three orders of magnitude smaller than necessary. A chloroplast is about
2 to 10 microns, so if you want to inject a whole chloroplast, you might want
to try a needle with a 10 micron inside diameter and perhaps a 20 micron
outside diameter.
To transform/fect (there really is no difference these days with the commonly used terminology) a chloroplast, you would need a needle smaller than your target.
Plant cells can be 100 microns or even 400 microns
across, and you might want to start with the big ones anyway, for practice.
While GalInStan (an alloy of gallium, indium, and tin that is non-toxic but liquid)
is fun to play with, I would bet that a rubber bulb from an eye dropper would
give you enough control, especially if you pushed it all the way down onto the
glass so that there was a lot less motion available. You could then try sucking
chloroplasts out of one cell and sticking them into another.
Why can't you just use water? It compresses too much???
It might be interesting to see what happens when you inject chloroplasts into
a plant like creeping dodder, which has lost chloroplasts as it evolved into a
parasite.
Another interesting project might be to inject C4 chloroplasts into C3 plants,
and see if they produce more sugar as a result. Sucking out all of the
Doesn't C4 require more energy because there is an extra layer that CO2 needs to get pumped through, by way of oxaloacetate and malate intermediates? The advantage of C4 carbon fixation is to retain moisture, by way of soaking CO2 into bundle sheath cells around a cell/chloroplast with RuBisCo in it, so the CO2:O2 ratio is higher when the stomata are closed to prevent evaporation loss. (97% water taken through roots is lost through evap in C3 plants)
--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
Mega
Nov 17, 2012, 4:31:58 PM11/17/12
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Hi,
as I'll be getting Agrobacterium plasmids quite soon,
and there was that paper that said chloroplast transformation using agrobacterium is feasible,
do you think it would be worth a try?
Basically, I'd use an empty Ti plasmid, PCR a kanamycin resistance and lux operon within it.
I think, I wouldn't even need a promoter etc. because the chloroplasts can express operons. So in some of the transformed chloroplasts the gene cassete is inserted correctly between two genes of an operon.
Am I completely wrong?
as I'll be getting Agrobacterium plasmids quite soon,
and there was that paper that said chloroplast transformation using agrobacterium is feasible,
do you think it would be worth a try?
Basically, I'd use an empty Ti plasmid, PCR a kanamycin resistance and lux operon within it.
I think, I wouldn't even need a promoter etc. because the chloroplasts can express operons. So in some of the transformed chloroplasts the gene cassete is inserted correctly between two genes of an operon.
Am I completely wrong?
Sebastian S. Cocioba
Nov 18, 2012, 2:38:27 AM11/18/12
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Agro injects into nucleus as far a I know. Normally plastid transformation is dun via gold gun. If you check the plos one article from the guy who made bioglow, it references his choice against agro for plastid transformation.
Sebastian S Cocioba
CEO & Founder
New York Botanics, LLC
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Cathal Garvey
Nov 19, 2012, 5:04:51 PM11/19/12
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By default, Agrobacterium is nuclear-targeting, but I think this is due
to targeting factors in the DNA of the transferred plasmid, or just
sheer chance of where the DNA ends up.
One way to get DNA to more reliably target alternative organelles is to
include DNA sequences matching the target sites of DNA binding proteins
found in said organelles, provided the proteins are made in the
cytoplasm. Essentially, you're hoping that as the cell finishes a
protein destined for the Chloroplast, your DNA will be bound by the
protein as the cell moves the protein into the organelle, dragging your
DNA in with it.
So, if your DNA contains valid promoter regions for chloroplasts, you'd
expect to see an uptick in chloroplast transfection already, provided
that some or all of the promoter-binding proteins are made in the
cytoplasm and not inside the chloroplast itself.
If that's not the case, you might have to find some DNA-binding
chloroplast proteins that *are* made in the cytoplasm (bearing in mind
that the genes coding for said proteins might be in the nucleus, not the
chloroplast genome), and shiv in a few sequences that might be bound by
those proteins.
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to targeting factors in the DNA of the transferred plasmid, or just
sheer chance of where the DNA ends up.
One way to get DNA to more reliably target alternative organelles is to
include DNA sequences matching the target sites of DNA binding proteins
found in said organelles, provided the proteins are made in the
cytoplasm. Essentially, you're hoping that as the cell finishes a
protein destined for the Chloroplast, your DNA will be bound by the
protein as the cell moves the protein into the organelle, dragging your
DNA in with it.
So, if your DNA contains valid promoter regions for chloroplasts, you'd
expect to see an uptick in chloroplast transfection already, provided
that some or all of the promoter-binding proteins are made in the
cytoplasm and not inside the chloroplast itself.
If that's not the case, you might have to find some DNA-binding
chloroplast proteins that *are* made in the cytoplasm (bearing in mind
that the genes coding for said proteins might be in the nucleus, not the
chloroplast genome), and shiv in a few sequences that might be bound by
those proteins.
www.indiebiotech.com
twitter.com/onetruecathal
joindiaspora.com/u/cathalgarvey
PGP Public Key: http://bit.ly/CathalGKey
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