Finally a chance to create a glowing plant?
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Mega
Feb 10, 2012, 10:14:48 AM2/10/12
to DIYbio
http://www.pgreen.ac.uk/a_pls_fr.htm - Agrobacterium disarmed Ti-
Plasmid
Kanamycin– Selektionsmarker (‘nptI’)
LacZ – Marker Blue-white screening
Here is a plasmid, as I understood this is a fully functional
Agrobacterium Ti-Plasmid.
It has a restriction site "SalI".
My pVIB has also SalI !!!
So ligating them together makes a plasmid that induces bioluminescense
in plants.
A question: The promotor works for agrobacterium and coli too?? Will
the bacteria glow too to mark them?
Because also the amp part of lux could be inserted and those will too
not make the X-Gal blue and be kanamycin resistant.
-> take glowing bacterium colony, culture them, plasmid preparation,
plasmid into void agrobacterium.
ready.
My plan is, going to a professor at mine, tell him and ask wheter I
could possibly use the lab and university as shiping adress.
Are my thoughts correct??
Yours
Plasmid
Kanamycin– Selektionsmarker (‘nptI’)
LacZ – Marker Blue-white screening
Here is a plasmid, as I understood this is a fully functional
Agrobacterium Ti-Plasmid.
It has a restriction site "SalI".
My pVIB has also SalI !!!
So ligating them together makes a plasmid that induces bioluminescense
in plants.
A question: The promotor works for agrobacterium and coli too?? Will
the bacteria glow too to mark them?
Because also the amp part of lux could be inserted and those will too
not make the X-Gal blue and be kanamycin resistant.
-> take glowing bacterium colony, culture them, plasmid preparation,
plasmid into void agrobacterium.
ready.
My plan is, going to a professor at mine, tell him and ask wheter I
could possibly use the lab and university as shiping adress.
Are my thoughts correct??
Yours
Cathal Garvey
Feb 10, 2012, 10:19:32 AM2/10/12
to diy...@googlegroups.com
Sadly, no: the pVib plasmid contains DNA from bacteria, which will only
function in bacteria. The promoters, ribosome-binding-sites and the
codon biases will all be entirely wrong for plants, I'm afraid.
function in bacteria. The promoters, ribosome-binding-sites and the
codon biases will all be entirely wrong for plants, I'm afraid.
In order to get it to work in plants, you'd need to re-jig the entire
operon into plant-compatible code, then put that into the Ti plasmid,
and then transfect plant cells with Agrobacteria carrying the gene.
Have you got glowing E.coli yet though? Because really that's the first
step! If you've got pVib on hand, and E.coli to work with, and a
licensed lab to work *in*, go do that and get your first transformation
done. Plants are hard work!
On 10/02/12 15:14, Mega wrote:
> http://www.pgreen.ac.uk/a_pls_fr.htm - Agrobacterium disarmed Ti-
> Plasmid
> Kanamycin� Selektionsmarker (�nptI�)
> LacZ � Marker Blue-white screening
>
>
> Here is a plasmid, as I understood this is a fully functional
> Agrobacterium Ti-Plasmid.
>
>
> It has a restriction site "SalI".
> My pVIB has also SalI !!!
>
> So ligating them together makes a plasmid that induces bioluminescense
> in plants.
>
> A question: The promotor works for agrobacterium and coli too?? Will
> the bacteria glow too to mark them?
> Because also the amp part of lux could be inserted and those will too
> not make the X-Gal blue and be kanamycin resistant.
> -> take glowing bacterium colony, culture them, plasmid preparation,
> plasmid into void agrobacterium.
> ready.
>
>
> My plan is, going to a professor at mine, tell him and ask wheter I
> could possibly use the lab and university as shiping adress.
>
> Are my thoughts correct??
>
>
> Yours
>
--
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Mega
Feb 10, 2012, 11:23:46 AM2/10/12
to DIYbio
Yeah, ok...
On Tuesday I'll do the pVIB - Coli.
(I've got a test on monday... So the next day I get a friend of mine
look how I do it and maybe help me and learn)
Will it work if I get a plasmid from fireflies??
___
that should work. With the ribosome-b-s you are surely right.
Codon-biases? I've had to read it from wikipedia. It slowes down gene
expression mainly. (But if it STOPS expression, didn't say that )
On 10 Feb., 16:19, Cathal Garvey <cathalgar...@gmail.com> wrote:
>
> In order to get it to work in plants, you'd need to re-jig the entire
> operon into plant-compatible code, then put that into the Ti plasmid,
> and then transfect plant cells with Agrobacteria carrying the gene.
>
> Have you got glowing E.coli yet though? Because really that's the first
> step! If you've got pVib on hand, and E.coli to work with, and a
> licensed lab to work *in*, go do that and get your first transformation
> done. Plants are hard work!
>
> On 10/02/12 15:14, Mega wrote:
>
>
>
>
>
>
>
>
>
> >http://www.pgreen.ac.uk/a_pls_fr.htm- Agrobacterium disarmed Ti-
On Tuesday I'll do the pVIB - Coli.
(I've got a test on monday... So the next day I get a friend of mine
look how I do it and maybe help me and learn)
Will it work if I get a plasmid from fireflies??
___
> Sadly, no: the pVib plasmid contains DNA from bacteria, which will only
> function in bacteria. The promoters, ribosome-binding-sites and the
> codon biases will all be entirely wrong for plants, I'm afraid.
The promotor of is provided by agrobacterium plasmid, isn't it??? So
> function in bacteria. The promoters, ribosome-binding-sites and the
> codon biases will all be entirely wrong for plants, I'm afraid.
that should work. With the ribosome-b-s you are surely right.
Codon-biases? I've had to read it from wikipedia. It slowes down gene
expression mainly. (But if it STOPS expression, didn't say that )
On 10 Feb., 16:19, Cathal Garvey <cathalgar...@gmail.com> wrote:
>
> In order to get it to work in plants, you'd need to re-jig the entire
> operon into plant-compatible code, then put that into the Ti plasmid,
> and then transfect plant cells with Agrobacteria carrying the gene.
>
> Have you got glowing E.coli yet though? Because really that's the first
> step! If you've got pVib on hand, and E.coli to work with, and a
> licensed lab to work *in*, go do that and get your first transformation
> done. Plants are hard work!
>
> On 10/02/12 15:14, Mega wrote:
>
>
>
>
>
>
>
>
>
> > Plasmid
> > Kanamycin Selektionsmarker ( nptI )
> > LacZ Marker Blue-white screening
> > Kanamycin Selektionsmarker ( nptI )
Mega
Feb 10, 2012, 11:25:20 AM2/10/12
to DIYbio
(I know fireflies don't have plasmids. But there will be plasmids
containing fire fly gene cassette?? ?? )
containing fire fly gene cassette?? ?? )
Patrik
Feb 10, 2012, 9:31:57 PM2/10/12
to DIYbio
On Feb 10, 8:25 am, Mega <masterstorm...@gmail.com> wrote:
> (I know fireflies don't have plasmids. But there will be plasmids
> containing fire fly gene cassette?? ?? )
For the firefly system, as I understand it the pathway to synthesize
> (I know fireflies don't have plasmids. But there will be plasmids
> containing fire fly gene cassette?? ?? )
the luciferin is not fully understood. So you can find plasmids that
can express the firefly luciferase enzyme in E. coli. But then you
have to add luciferin separately to make it light up. (The plasmid may
also carry a gene to recycle the oxidized luciferin, so you don't need
to keep adding luciferin.)
Vibrio is probably your best bet for now, since it has both the
luciferin synthesis pathway, and the luciferase genes on one
convenient plasmid. We're actually ordering an educational kit with
BioCurious right now, as part of our new community project on
bioluminescence. We figured we'd just walk through the kit first to
get everyone up to speed, and then start tinkering with it:
http://www.modernbio.com/experiment/producing-strain-e-coli-glows-dark
(They only sell to schools, unfortunately.)
Mega
Feb 11, 2012, 4:16:14 AM2/11/12
to DIYbio
> http://www.modernbio.com/experiment/producing-strain-e-coli-glows-dark
>
> (They only sell to schools, unfortunately.)
It's kind of cheaper with Carolina Biological Supply .... To private
persons they only send the plasmid, not the transformation kit.
9.80$ or so... (Although frozen shipping is extremely expensive...)
In your link they provide you with both amp-agar and pure
ampicillin...
Pat
Feb 11, 2012, 7:37:11 AM2/11/12
to DIYbio
This kit should have all the tools you need for transforming bacteria:
http://brantford.kijiji.ca/c-buy-and-sell-toys-games-New-in-box-Science-Wiz-DNA-Wizard-Kit-Retail-34-99-W0QQAdIdZ218440808
http://brantford.kijiji.ca/c-buy-and-sell-toys-games-New-in-box-Science-Wiz-DNA-Wizard-Kit-Retail-34-99-W0QQAdIdZ218440808
Mega
Feb 11, 2012, 10:10:52 AM2/11/12
to DIYbio
Cathal, how sure are you that the bacterial lux won't be able to be
expressed?
Do you have an idea what's the chance? 0% 10% 30% (50%) ??
If it's 1% or below it wouldn't be worth doing research on this for
me.
But I think, maybe different plants have slightly different promoters/
coding/ condon biases...
Maybe ferns are more related to bacteria than (say) marbles etc.
Once havinbg those agrobacteria I could try to infect all kinds of
plants.
Not a chance??
If you assume the chance to about 30% I think it would be worth giving
it a try (to ask my proff. to be allowed to try it in the lab)
expressed?
Do you have an idea what's the chance? 0% 10% 30% (50%) ??
If it's 1% or below it wouldn't be worth doing research on this for
me.
But I think, maybe different plants have slightly different promoters/
coding/ condon biases...
Maybe ferns are more related to bacteria than (say) marbles etc.
Once havinbg those agrobacteria I could try to infect all kinds of
plants.
Not a chance??
If you assume the chance to about 30% I think it would be worth giving
it a try (to ask my proff. to be allowed to try it in the lab)
Cathal Garvey
Feb 12, 2012, 5:01:26 AM2/12/12
to diy...@googlegroups.com
The odds of it expressing are virtually nil, I'm afraid.
It's mostly due to the promoter structure being *entirely* different,
though the ribosome binding sites and codon bias will also be all wrong.
Plants are just not bacteria; they've been separated by eons of evolution.
If you can get the DNA into the chloroplasts, or even the mitochondria,
then you might have a more-than-1%-chance. But it'd still be a pretty
small chance of it working right away: Chloroplasts and Mitochondria are
pretty distantly related to Vibrio species.
Patrik
Feb 12, 2012, 5:20:51 AM2/12/12
to DIYbio
> If you can get the DNA into the chloroplasts, or even the mitochondria,
> then you might have a more-than-1%-chance.
That's an interesting thought. How would you even target something to
> then you might have a more-than-1%-chance.
the chloroplasts or mitochondria though?
Hm... looks like gene guns can transform organelles...
Cathal Garvey
Feb 12, 2012, 5:30:29 AM2/12/12
to diy...@googlegroups.com
You can do it for some species with a PEG-mediated method, too, but your
milage will vary by species and precise protocol.. Also, I think the
cells have to be digested into protoplasts, which calls for cellulase at
least; hard enzyme to find at home, right now.
milage will vary by species and precise protocol.. Also, I think the
cells have to be digested into protoplasts, which calls for cellulase at
least; hard enzyme to find at home, right now.
Nice thing about transforming chloroplasts of lower plants, at least, is
that you can select for transformants with a range of normal antibiotics
such as ampicillin. Of course, an antibiotic-free method would be nice.
To enhance specificity for the chloroplasts, you could incorporate a
chloroplast-only promoter sequence to your DNA. For nuclear delivery,
adding a promoter region for a constitutive transcription factor
sometimes enhances transformation efficiency, presumably because
proteins assembled in the cytoplasm catch the DNA on their way toward
the nucleus.
For that to work, you'd have to find a chloroplast DNA-binding protein
that is coded for in the nucleus and transported to the chloroplast once
finished; that subset of proteins have a chance of pulling DNA in with them.
Pat
Feb 12, 2012, 7:45:03 AM2/12/12
to DIYbio
Mega
Feb 12, 2012, 8:42:42 AM2/12/12
to DIYbio
How do you get the transformed chloroplasts into a living cell??
Using the PEG-Methode, is there a possibility that it gets anyhow into
the chloroplasts directly without additional help??
If the chance is 1:100, I'll do it 100 times.
Why use protoplasts when you could youse also seemen or pollen??
Using the PEG-Methode, is there a possibility that it gets anyhow into
the chloroplasts directly without additional help??
If the chance is 1:100, I'll do it 100 times.
Why use protoplasts when you could youse also seemen or pollen??
Patrik
Feb 13, 2012, 3:35:40 AM2/13/12
to DIYbio
Cathal Garvey
Feb 13, 2012, 5:40:16 AM2/13/12
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YES
Cathal Garvey
Feb 13, 2012, 5:55:09 AM2/13/12
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Boom, science. Great rundown on state of the art, thanks!
There's information in there on the PEG-mediated method; it's tricky by
the look of it. You have to make protoplasts, yes, but cells only
survive so long in the PEG medium before dying of it, so timing is
probably pretty important.
Also, because there are plenty of chloroplasts in the cell, and each has
plenty of genome copies, it's apparently challenging to make certain
you've got universally-transformed chloroplasts. I imagine to get
reliable transformation, you'd need a system that actively targets
non-transformed chromosomes for homologous recombination with the
transformed chromosomes, so that the chromosomes eventually all get a
copy of the new gene cassette.
For tidyness' sake, you'd also want this recombination system and any
selection genes in a self-excising cassette that you can trigger once
you're sure of your transformants' integrity.
I was surprised to see that protein yield from plastid transformants can
be very high; this would be very difficult to achieve, but probably
pretty rewarding!
Mega
Feb 17, 2012, 3:41:49 PM2/17/12
to DIYbio
http://en.wikipedia.org/wiki/Nostoc_pruniforme
That's cool...
A sphere formed by cyanobacteria.
Cyanobacteria are bacteria, so they should be able to express
plasmids.
I think that could be one of the easiest way to create 'kind-of-
plants' that glow.
That's cool...
A sphere formed by cyanobacteria.
Cyanobacteria are bacteria, so they should be able to express
plasmids.
I think that could be one of the easiest way to create 'kind-of-
plants' that glow.
Mega
Feb 21, 2012, 4:35:15 AM2/21/12
to DIYbio
Recently I saw this:
http://www.mykonet.ch/Leuchtpilze.htm
The I googled, and saw that there are kits available to grow Panellus
Stipticus (glowing mushroom) at home.
I was thinking how scientists could get a gene casette out of a
chromosom into a plasmid. Haven't read anything about that before, but
I think you would do it as following (?):
The genome has to be sequenced. You know which restriction enzyme cuts
what sequence and around the lux operon you search for such a target.
Then the enzyme will cut the chromosomes into pieces. You know, how
much base-pairs the lux operon has and do electrophoresis. So the
genes seperate and you take your lux operon, do a PCR and cut it into
a pUC18 or something??
(I think the genome has been sequenced because of that paragraf of
wikipedia: )
"Using techniques of genetic complementation, Macrae paired
nonluminescent monocaryons with luminescent ones, and concluded that
luminosity in P. stipticus is an inherited character, and governed by
a single pair of alleles in which luminosity was dominant over
nonluminosity. Luminosity factors were independent of intersterility
factors"
http://www.mykonet.ch/Leuchtpilze.htm
The I googled, and saw that there are kits available to grow Panellus
Stipticus (glowing mushroom) at home.
I was thinking how scientists could get a gene casette out of a
chromosom into a plasmid. Haven't read anything about that before, but
I think you would do it as following (?):
The genome has to be sequenced. You know which restriction enzyme cuts
what sequence and around the lux operon you search for such a target.
Then the enzyme will cut the chromosomes into pieces. You know, how
much base-pairs the lux operon has and do electrophoresis. So the
genes seperate and you take your lux operon, do a PCR and cut it into
a pUC18 or something??
(I think the genome has been sequenced because of that paragraf of
wikipedia: )
"Using techniques of genetic complementation, Macrae paired
nonluminescent monocaryons with luminescent ones, and concluded that
luminosity in P. stipticus is an inherited character, and governed by
a single pair of alleles in which luminosity was dominant over
nonluminosity. Luminosity factors were independent of intersterility
factors"
Mega
Jul 13, 2012, 4:05:14 PM7/13/12
to diy...@googlegroups.com
Once again, this old topic :D
I'm gonna get that DNA synthesized, but first I have to go haunting for fundings.... If you say it will work. It will cost some 3800$ so I have to be sure it works.
The bacterial luciferin-luciferase system per se should work in plants. (Why not?)
If I have it synthesized like this: best viral Plant promotor known - RBS - LuxA (codon usage modified!) - Terminator --- same for Lux B,C,D,E.... it will definitely create the substances needed to glow in planta???
The company will insert it into any vector, so I choose an agrobacterium Ti-Plasmid.
Insert Plasmids (binary system) into agrobacterium. Put it on plant seemen. Goal - a glowing plant??
I'm gonna get that DNA synthesized, but first I have to go haunting for fundings.... If you say it will work. It will cost some 3800$ so I have to be sure it works.
The bacterial luciferin-luciferase system per se should work in plants. (Why not?)
If I have it synthesized like this: best viral Plant promotor known - RBS - LuxA (codon usage modified!) - Terminator --- same for Lux B,C,D,E.... it will definitely create the substances needed to glow in planta???
The company will insert it into any vector, so I choose an agrobacterium Ti-Plasmid.
Insert Plasmids (binary system) into agrobacterium. Put it on plant seemen. Goal - a glowing plant??
Adam Levine
Jul 13, 2012, 4:09:40 PM7/13/12
to diy...@googlegroups.com
I'd love to be on your test grower list - We operate a small native nursery and would love to devote space to truly new genetics
Adam B. Levine
http://MindToMatter.org
http://MindToMatter.org
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Cathal Garvey
Jul 13, 2012, 5:34:06 PM7/13/12
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> so I have to
> be sure it works.
Here's your problem. There is _no_ way to know whether it will work as
> be sure it works.
intended until you try it.
Biology's complex; while we can often make pretty good predictions about
qualitative stuff: "will it glow, or will it not glow?".. we're pretty
much in the dark on the quantitative stuff: "How much will it glow? Will
it even be perceptible?"
You have two problems; without someone else doing it first, you don't
know if it'll work _at all_: the proteins might not end up in the
correct cell compartments, the precursors might get depleted too
quickly, the genes might get silenced by methylation.. etc.
Further, you don't know whether it will glow brightly enough to be visible.
So, if you wanna do it (it's ambitious, it's awesome!), be prepared to
not know if it'll work.
Andreas Sturm
Jul 14, 2012, 7:42:45 AM7/14/12
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> the genes might get silenced by methylation.. etc.
Ok, But that can depend on the plant?? If I (actually, my agrobacterium) put it into a willow, it may get silenced. Then I put it into a maple, and it may work?And: the chemicals needed (same chemicals as in bacteria) will be produced? In what quantities we don't know. Clearly.
>Further, you don't know whether it will glow brightly enough to be visible.
You could add aldehydes (such as decanal) to the water you 'feed' it with. Because the aldehydes will be the limiting factor.
Then it will glow brighter. If the luciferin works (which sould work anyway).
2012/7/13 Cathal Garvey <cathal...@gmail.com>
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Andreas Sturm
Jul 14, 2012, 8:48:11 AM7/14/12
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http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=Search&doptcmdl=Citation&defaultField=Title%20Word&term=Olsson[author]%20AND%20Engineering%20of%20monomeric%20bacterial%20luciferases%20by%20fusion%20of%20luxA%20and%20luxB%20genes%20in%20Vibrio%20harveyi.
Does that imply that lux A synthesis and then lux B synthesis won't work in planta, but you must fuse them?
Does that imply that lux A synthesis and then lux B synthesis won't work in planta, but you must fuse them?
2012/7/14 Andreas Sturm <masters...@gmail.com>
doctor.perkins
Jul 14, 2012, 9:26:05 AM7/14/12
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Here's a website I found recently that may be of interest. IdeaConnection Crowdfunding is a service for creative scientists, engineers, innovators and inventors to raise funds in order to do research, to obtain a provisional patent or a patent, to fund a startup, or to build an invention. It says that last year, crowdfunding projects raised $1.5 billion.
Adam Levine
Jul 14, 2012, 10:31:15 AM7/14/12
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It should be noted that ideaconnection is soliciting for their initial round of projects to seek funding (i.e. they haven't proven this works yet) - The 1.5 billion Crowdfunding number is misleading because they're using the entire industry number in a market where the top nexus gets 80%+ of the attention/funding and the other 40 fight over the remaining 20%. For the time being, that would be Kickstarter.com.
And Kickstarter could be used for this, I supported this project to great results for a guy who wanted to develop a autonomous algae based biodiesel generating wavepowered robot, sought $2,000 offering rather weaksauce rewards and still scooped about 8000 in funding.
http://www.kickstarter.com/projects/1579413688/biofuel-for-everyone?ref=users
My wife and I are actually consulting on a few projects seeking crowdfunding, if anybody wants to talk about fundraising opportunities and the best way to present your idea to that audience, I'd love to help (gratis)
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Andreas Sturm
Jul 15, 2012, 5:12:05 AM7/15/12
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Kickstarter doesn't work for non-US citizens. I asked them, they said they'd be working on this.
Now I'm gonna start an Indiegogo campaign. Am quite ready to start it.
Now I'm gonna start an Indiegogo campaign. Am quite ready to start it.
2012/7/14 Adam Levine <adamlevi...@gmail.com>
Omri Drory
Jul 16, 2012, 2:34:08 AM7/16/12
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Hi Guys, We at Genome Compiler thought for some time to do a kickstarter project for glowing plants as a demonstration for the good that can come with synthetic biology and DIYbio attitude. We're in the process of setting the project out and hopefully our credibility will allow us to raise the money. We plan to use the proceeding to design and order the DNA and collaborate (with the money we raise) with the bio luminescence group at BioC (and possibly other groups like the 2010 Cambridge iGem team and JBEI).
We want to demonstrate how one can start with modest crowd funding source, free design software (genome compiler) and a community lab to create real impactful, beautiful living things. I hope this community will support and join our effort.
I'm also playing with the idea of approaching james cameron (from avatar fame) - he's a friend of peter diamandis from Singularity University (we're a SU company, Peter referred us to Google solve for < X > conference).
Will keep you updated.
Omri
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Omri Drory
Jul 16, 2012, 2:36:55 AM7/16/12
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Hi Mega, let's talk (om...@genomecompiler.com) - we might be able to order the DNA for you as part of our project.
Omri
Omri Drory
Jul 16, 2012, 2:52:10 AM7/16/12
to diy...@googlegroups.com
p.s - check this out: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0015461
On Friday, February 10, 2012 5:14:48 PM UTC+2, Mega wrote:
On Friday, February 10, 2012 5:14:48 PM UTC+2, Mega wrote:
http://www.pgreen.ac.uk/a_pls_fr.htm - Agrobacterium disarmed Ti-
Plasmid
Kanamycin– Selektionsmarker (‘nptI’)
LacZ – Marker Blue-white screening
Here is a plasmid, as I understood this is a fully functional
Agrobacterium Ti-Plasmid.
It has a restriction site "SalI".
My pVIB has also SalI !!!
So ligating them together makes a plasmid that induces bioluminescense
in plants.
A question: The promotor works for agrobacterium and coli too?? Will
the bacteria glow too to mark them?
Because also the amp part of lux could be inserted and those will too
not make the X-Gal blue and be kanamycin resistant.
-> take glowing bacterium colony, culture them, plasmid preparation,
plasmid into void agrobacterium.
ready.
My plan is, going to a professor at mine, tell him and ask wheter I
could possibly use the lab and university as shiping adress.
Are my thoughts correct??
Yours
Gavin Scott
Jul 16, 2012, 12:49:01 PM7/16/12
to diy...@googlegroups.com
Since, as I understand it, the luciferin-luciferase reaction requires oxygen (I just read that fireflies glow by producing nitric oxide to temporarily prevent the mitochondria from consuming all the oxygen), might you not end up with a situation where your plants glow, but only in direct sunlight where photosynthesis produces a surplus of O2?
G.
Nathan McCorkle
Jul 16, 2012, 1:57:53 PM7/16/12
to diy...@googlegroups.com
On Mon, Jul 16, 2012 at 2:52 AM, Omri Drory <om...@genomecompiler.com> wrote:
> p.s - check this out:
> http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0015461
>
It's a step in the right direction, but the control system isn't right
> p.s - check this out:
> http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0015461
>
"exposures 5 min"...
--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
Cathal Garvey
Jul 18, 2012, 10:00:18 AM7/18/12
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Do remember that bacterial bioluminescence and firefly bioluminescence,
and most other systems besides, are prime examples of _convergent
evolution_. They mostly bear no actual homology to one another.
That caveat shared however, you are right nonetheless: Bacterial
bioluminescence also requires and consumes oxygen. In plants, this
probably means you'll see best results under warm conditions with plenty
of water, which will encourage leaf stomata to open and allow free gas
exchange. You may find that plants in less ideal conditions will suffer
from oxygen limitation at night-time if they have to keep their stomata
closed to conserve water, and this might affect growth or survival.
Worth remembering when people raise the spectre of wild escapee plants,
I guess; there's no real advantage, and significant potential
disadvantages to bioluminescent plants in the wild.
and most other systems besides, are prime examples of _convergent
evolution_. They mostly bear no actual homology to one another.
That caveat shared however, you are right nonetheless: Bacterial
bioluminescence also requires and consumes oxygen. In plants, this
probably means you'll see best results under warm conditions with plenty
of water, which will encourage leaf stomata to open and allow free gas
exchange. You may find that plants in less ideal conditions will suffer
from oxygen limitation at night-time if they have to keep their stomata
closed to conserve water, and this might affect growth or survival.
Worth remembering when people raise the spectre of wild escapee plants,
I guess; there's no real advantage, and significant potential
disadvantages to bioluminescent plants in the wild.
Adam Levine
Jul 18, 2012, 10:27:28 AM7/18/12
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Any ideas on what kind of plant to try this with? If oxygen is a concern, I'd suggest using a low pressure aeroponics system - You can build one for about $80 at a hardware store.
Plants grown in this method do not use a substrate, rather being grown in little net pots that let their roots hang in dark air below. Under low pressure aeroponics, you basically have a recirculating nutrient solution stored in a reservoir. A variable timer (one minute on, several minutes off) controls a pump in the solution which when activated forces the solution through a PVC sprayer assembly, comprehensively wetting the roots of the plant(s). The pump turns off, the sprayed solution drains back into the reservoir.
This is ideal for many types of plants because it keeps the roots comprehensively wetted, but gives them regular and frequent "drying times' that encourage root growth as well as this whole type of system providing very granular control over the conditions your plant is subjected to.
This could be done indoors or outdoors, but if you wanted to control stomata opening/closing you could do that internally with a controlled humidifier.
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Andreas Sturm
Jul 18, 2012, 2:11:48 PM7/18/12
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I read something from NASA regarding this:
Under low pressure, the plant thinks it loses too much water and gets stressed. (Mars base will have significantly lower pressure inside)
On the other hand, more pressure makes a contrary effect.
A willow is a good plant because it is really into water. It grows best near rivers, it's just like weeds...
Under low pressure, the plant thinks it loses too much water and gets stressed. (Mars base will have significantly lower pressure inside)
On the other hand, more pressure makes a contrary effect.
A willow is a good plant because it is really into water. It grows best near rivers, it's just like weeds...
2012/7/18 Adam Levine <adamlevi...@gmail.com>
Adam Levine
Jul 18, 2012, 8:17:05 PM7/18/12
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I think you've misunderstood my use of "low pressure" - That's referring to the PSI of the pump and sprayer array compared to a "high pressure" system that uses a highly pressurized atomizer to turn the nutrient solution into such a fine mist that it passes through the outer membrane of the plant without actually wetting it.
You're talking about atmospheric pressure I believe(?), which is not something I've addressed.
Mega
Jul 19, 2012, 4:36:11 AM7/19/12
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Yeah, but it affects the oxygen uptake which was the ''source idea''.
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Mega
Oct 21, 2012, 6:46:46 AM10/21/12
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I came across somthing:
If I make the plant nucleus produce the bacterial luciferase proteins, they won't get methylated but glycolisated. The LuxAB have been shown to woork, but what about the other proteins? Won't they maybely work?
If I make the plant nucleus produce the bacterial luciferase proteins, they won't get methylated but glycolisated. The LuxAB have been shown to woork, but what about the other proteins? Won't they maybely work?
Cathal Garvey
Oct 22, 2012, 5:31:19 AM10/22/12
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It's certainly not something I know a lot about, but glycosylation
usually occurs on pseudo-motifs in the peptide sequence of the protein,
I think. So, if there aren't any obvious glycosylation sites in your
proteins, you're probably ok.
Of course, these sites can occur by accident, and if the glycosylation
blocks the active site or regulatory sites of the enzyme, perhaps it'd
screw things up; you might have to guesstimate from uniprot data whether
or not this is likely and perhaps risk editing an amino here and there
to make glycosylation less likely. Far enough from the active site, if
you just replace aminos with very similar relatives, you will probably
be ok. Be careful of aminos that have very odd bond angles on the amino
backbone, because they are likely to have structural significance; I
think proline comes to mind? Or was that glycine? :)
usually occurs on pseudo-motifs in the peptide sequence of the protein,
I think. So, if there aren't any obvious glycosylation sites in your
proteins, you're probably ok.
Of course, these sites can occur by accident, and if the glycosylation
blocks the active site or regulatory sites of the enzyme, perhaps it'd
screw things up; you might have to guesstimate from uniprot data whether
or not this is likely and perhaps risk editing an amino here and there
to make glycosylation less likely. Far enough from the active site, if
you just replace aminos with very similar relatives, you will probably
be ok. Be careful of aminos that have very odd bond angles on the amino
backbone, because they are likely to have structural significance; I
think proline comes to mind? Or was that glycine? :)
Andreas Sturm
Oct 22, 2012, 7:38:10 AM10/22/12
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Ok, well, but if the stuff gets codon-optimized by the company after your editing, you may get problems here...
Cathal Garvey
Oct 24, 2012, 10:31:06 AM10/24/12
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Codon optimisation doesn't change the amino acid sequence, and it's the amino acid sequence that signals glycosylation systems etc.
On 22 October 2012 12:38, Andreas Sturm <masters...@gmail.com> wrote:
Ok, well, but if the stuff gets codon-optimized by the company after your editing, you may get problems here...
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Andreas Sturm
Oct 24, 2012, 12:02:53 PM10/24/12
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Ah, that's true!
(What did I think of?)
(What did I think of?)
Glucoperon
Nov 5, 2013, 10:57:13 PM11/5/13
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Yo Mega! how far did you get on this project???
Mega [Andreas Stuermer]
Nov 18, 2013, 8:19:41 AM11/18/13
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Sorry for replying so late. Didn't see it.
The chinese are synthesizing the regulatory sequences for me, and after some negotiation they even do a PCR of lux and kanR for quie little money.
As soon as they are ready, I get a ready to use pGlowroplast! As said, I will share it with anybody who is interrested. No IP issues with the version in the chinese vector. The version in pGreenII-0000 can't share without permission of JIC
Emerick Larkin
Nov 19, 2013, 11:20:53 PM11/19/13
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Awesome, keep us updated !
Andreas Stuermer
Nov 20, 2013, 3:28:52 AM11/20/13
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I will ;)
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