*experienced* beginner is creating a bioluminescent plant - both synbio and old-fashioned way

[Mega]

Hi everyone. You may remember the old thread with a similar name, *some* time ago :D

https://groups.google.com/forum/?fromgroups=#!topic/diybio/MhOcaOT09lA

 

 

There's news.

 

As you surely have heard, Genome Compiler is launching a kickstarter- crowdfunding campaign (very soon), to raise money and have DNA synthesized that will be put into plant (synthetic constructs for both nucleus / chloroplast) and makes it glow in the dark. (Everyone can place designs and the best ones will be elected by a comitee and synthesized. See http://www.glowingplant.com/ for additional info). Gonna post the link to kickstarter as soon as it is online. You gotta look at their video - it's awesome!

 

We at the Ars Electronica Biolab will be under the lucky ones who will get the synDNA then. Also BioCurious and other interested labs get the DNA.

 

 

 

Additionally, we got the idea to use the meantime to do an old-fashioned, albeit  long-winded, approach: Do a PCR of a chloroplast promoter, of RBS + Lux operon, of Kanamycin resistance, of a terminator and clone it into pGreenII. I can/will also post the sequences, however if one wants to make an *awesome* glowing  plant, getting the DNA from Genome Compiler would be more advisable...(The primers alone we’ll order cost more than 50 Euros, while one gets glowing plant seeds as a gift for donating 40$ to the kickstarter campaign.)

We would put that insert into pGreenII, pGreenII into Agrobacterium (containing also pSoup), and place them on a plant leave piece -> regenerate a plantlet out of it on selective medium.

According to this http://www.ncbi.nlm.nih.gov/pmc/articles/PMC554353/  paper, Chloroplast transformation by Agrobacterium works. However, there is not a single other paper having done that a second time.

Now we are curious to see, if that is really true.

 

 

 

 

Why would we do an additional hard-work, old fashioned restriction-ligation approach while also getting the synthetic DNA, which - by the way - will also glow much stronger, due to better promoters, optimal 5' and 3' UTR, due to genes from other species enhancing light output e.g. FMNH availability, antenna proteins enhancing light output dramatically by shifting the light into less-energetic wavelengths (less-energetic wavelength -> more photons, better visible), .... ?

 

I guess the simple answer is curiosity. And is there a better waste of time than ligate together DNA pieces for a glowing plant (also when just weakly glowing)? :D See if we can do the restriction-ligation of four PCR products, select for transformants the "annoying" way by X-Gal. Then see if the agrobacterium-plastid transformation paper is true (But why should De Block et al. lie to us? :D ). And if that works,  whether the transplastomic plants will glow strong enough to be seen with naked eye in the darkness after all...

(Here was also mentioned Dr. Krichevski once, who inserted a lux-operon into chloroplasts, which was/is visible to the naked eye, but only after 5 minutes of eye-adaption)

 

But, If the synthetic DNA arrives before we got the old-fashioned approach ready, it will be paused, and perhaps/probably even cancelled.

You see, the synthetic DNA is even more exciting, because it is designed to be worlds brighter, especially because you don’t have limits when designing, don’t need to design extra primers for each gene, get a template, …  Last but not least: no error-prone restriction work

 

So, in best case, we‘ll have two plants, one labeled “traditional genetic engineering” , weakly blue glowing and one “synthetic DNA” – which will hopefully be very bright yellow or green or red, well be seen without eye-adaption. Hopefully bright enough to read a book when laying under it - but I'm positive ;)

 

 

Maybe (probably :D ) there will be some detail-related questions… ;) Like how to get the chloroplast DNA from a leave, does it need clean-up before PCR, etc. :)

We thought of just mechanically damaging a leave piece and then 90°C for 20 minutes... 

Best,

Andreas

 

[Mega]

Reposting it: 

The kickstarter project is open!

http://www.kickstarter.com/projects/antonyevans/glowing-plants-natural-lighting-with-no-electricit

13.500$ donated in one hour !!

[Dakota Hamill]

Pretty crazy. It'll be interesting to see how it all pans out! $40
for your own transgenic glowing plant sounds pretty sweet to me.

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[Mega]

Yeah, like I said... 

40$ and you get the ready, bright glowing plant seeds   -  against 53 Dollars for primer synthesis alone. Without guarantee that all your restriction work will work. A bargain.

[Patrik D'haeseleer]

Hi Mega!

Now that the cat is officially out of the bag - looking forward to working with you!

Did you wind up submitting anything for the Genocon promoter design challenge a while back? I think we got about 10 different designs in, mainly for dark-expressing promoters - should come in handy for the glowing plant project.

Patrik

[Brian Degger]

any idea of the legality in the eu, which is more of a process9gm) than product(edible) kind of place. 

cheers

Brian

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----------------------------------------

[Thomas Landrain]

I can already answer you Brian. There are no GMO plants allowed in Europe if it is not for animal food. We'll probably wait for a fair amount of time before seeing luminescent trees sprouting in Europe...

[Mega]

Hi Patrik,
Didn't design any plant promoters yet. I was just using the strongest natural constitutive promoter (viral) .... The first try I would make it consitutive, as bright as possible,




In Europe you can cultivate them in a S1 lab of course ;) And maybe: go to court fight for my right to cultivate a glowing plant in my house :D Regarding that in one design the plant has glowing chloroplasts, and chloroplasts are not spread by pollen.

[Pieter]

We might not be allowed to release it, but growing in a contained place is doable.

In the museum here in The Hague we now have Adam Zaretsky's Errarium on display, hosting GMO zebrafish injected with algae. A GMO permit has been filed. Basically it's a box in a box in a box. http://ja-natuurlijk.com/site2/category/kunstenaars-en

[Brian Degger]

So, more gmo showroom :(

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[Cathal Garvey (Phone)]

If the glowing plant works, they could sell the seeds as animal feed to EU and we could cultivate from that.. ;)

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[Andreas Sturm]

GMO feeding animals is not illegal?

In my country, it is not allowed if it contains more than 1% GM I think...

But great idea...

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[Cathal Garvey]

-----BEGIN PGP SIGNED MESSAGE-----
Hash: SHA256

Nope, that's only for *people*. You can feed animals as much GMO seed
as you like, as long as you didn't grow it in the EU.

It's one of the finer points of hypocrisy against GMOs in Europe, that
we're the only domestic animal in the Union not already regularly
eating GMOs.

On 04/24/2013 02:52 PM, Andreas Sturm wrote:
> GMO feeding animals is not illegal?
>
> In my country, it is not allowed if it contains more than 1% GM I
> think...
>
>
> But great idea...
>
>
> On Wed, Apr 24, 2013 at 11:08 AM, Cathal Garvey (Phone)
> <cathal...@cathalgarvey.me

> <mailto:cathal...@cathalgarvey.me>> wrote:
>
> If the glowing plant works, they could sell the seeds as animal
> feed to EU and we could cultivate from that.. ;)
>
>
> Brian Degger <brian....@gmail.com

> <mailto:brian....@gmail.com>> wrote:
>
> So, more gmo showroom :(
>
> On 24 Apr 2013 08:26, "Pieter" <pieterva...@gmail.com

> <mailto:pieterva...@gmail.com>> wrote:
>
> We might not be allowed to release it, but growing in a contained
> place is doable.
>
> In the museum here in The Hague we now have Adam Zaretsky's
> Errarium on display, hosting GMO zebrafish injected with algae. A
> GMO permit has been filed. Basically it's a box in a box in a box.
> http://ja-natuurlijk.com/site2/category/kunstenaars-en

> <http://ja-natuurlijk.com/site2/category/kunstenaars-en/#content-kunstnaar-0>

>
>
>
> On Wednesday, 24 April 2013 08:12:39 UTC+2, Mega wrote:
>
> Hi Patrik, Didn't design any plant promoters yet. I was just using
> the strongest natural constitutive promoter (viral) .... The first
> try I would make it consitutive, as bright as possible,
>
>
>
>
> In Europe you can cultivate them in a S1 lab of course ;) And
> maybe: go to court fight for my right to cultivate a glowing plant
> in my house :D Regarding that in one design the plant has glowing
> chloroplasts, and chloroplasts are not spread by pollen.
>
>
>
>
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[Nathan McCorkle]

The new wave of euroamerican immgration, not sparked by a potato famine or men wearing kilts, but a science freedom famine :P

(or is there a society more *free* than the U.S to do DIYbio/startup-biotech?)

-Nathan

[Cathal Garvey]

-----BEGIN PGP SIGNED MESSAGE-----
Hash: SHA256

Lots, I suspect, although the sentiment on the ground won't be as
friendly as the legal end. I imagine there are plenty of countries
that haven't even bothered updating their laws in decades and were
never part of prohibitionist frameworks like the EU.

On 04/24/2013 07:47 PM, Nathan McCorkle wrote:
> The new wave of euroamerican immgration, not sparked by a potato
> famine or men wearing kilts, but a science freedom famine :P
>
> (or is there a society more *free* than the U.S to do
> DIYbio/startup-biotech?)
>
>
> On Wed, Apr 24, 2013 at 8:01 AM, Cathal Garvey
> <cathal...@cathalgarvey.me

> <mailto:cathal...@cathalgarvey.me>> wrote:
>
> Nope, that's only for *people*. You can feed animals as much GMO
> seed as you like, as long as you didn't grow it in the EU.
>
> It's one of the finer points of hypocrisy against GMOs in Europe,
> that we're the only domestic animal in the Union not already
> regularly eating GMOs.
>
> On 04/24/2013 02:52 PM, Andreas Sturm wrote:
>> GMO feeding animals is not illegal?
>
>> In my country, it is not allowed if it contains more than 1% GM
>> I think...
>
>
>> But great idea...
>
>
>> On Wed, Apr 24, 2013 at 11:08 AM, Cathal Garvey (Phone)
>> <cathal...@cathalgarvey.me
>> <mailto:cathal...@cathalgarvey.me>

>> <mailto:cathal...@cathalgarvey.me

> <mailto:cathal...@cathalgarvey.me>>> wrote:
>
>> If the glowing plant works, they could sell the seeds as animal
>> feed to EU and we could cultivate from that.. ;)
>
>
>> Brian Degger <brian....@gmail.com

>> <mailto:brian....@gmail.com> <mailto:brian....@gmail.com

>> <mailto:brian....@gmail.com>>>
> wrote:
>
>> So, more gmo showroom :(
>
>> On 24 Apr 2013 08:26, "Pieter" <pieterva...@gmail.com
> <mailto:pieterva...@gmail.com>

>> <mailto:pieterva...@gmail.com

> <mailto:pieterva...@gmail.com>>> wrote:
>
>> We might not be allowed to release it, but growing in a
>> contained place is doable.
>
>> In the museum here in The Hague we now have Adam Zaretsky's
>> Errarium on display, hosting GMO zebrafish injected with algae.
>> A GMO permit has been filed. Basically it's a box in a box in a
>> box. http://ja-natuurlijk.com/site2/category/kunstenaars-en
>
> <http://ja-natuurlijk.com/site2/category/kunstenaars-en/#content-kunstnaar-0>
>
>
>
>
>> On Wednesday, 24 April 2013 08:12:39 UTC+2, Mega wrote:
>
>> Hi Patrik, Didn't design any plant promoters yet. I was just
>> using the strongest natural constitutive promoter (viral) ....
>> The first try I would make it consitutive, as bright as
>> possible,
>
>
>
>
>> In Europe you can cultivate them in a S1 lab of course ;) And
>> maybe: go to court fight for my right to cultivate a glowing
>> plant in my house :D Regarding that in one design the plant has
>> glowing chloroplasts, and chloroplasts are not spread by pollen.
>
>
>
>
>> -- -- You received this message because you are subscribed to
>> the Google Groups DIYbio group. To post to this group, send email
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> options,
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>
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>

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[Mega]

Newsupdate... 

Included this gene into a design which is native to many plant species.... http://journals2005.pasteur.ac.ir/NB/23(9)/1177%20-%201180.pdf

Confers resistance to kanamycin when overexpressed. Does not work in bacteria. 

 

Could you call such a glowing plant "cisgenic" instead of transgenic ? Because it is a native plant gene  (and as a marker not GFP but Lux - but both just optical markers, used many times, not toxic)

[Cathal Garvey]

-----BEGIN PGP SIGNED MESSAGE-----
Hash: SHA256

Nope. "Cisgenic" means "from same species", although some liberal
interpretations say "same genus". Assume the former. So unless it's
A.thaliana DNA into another strain/cultivar of A.thaliana, it's
transgenic.

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[Nathan McCorkle]

Cathal, the paper mentions it IS an A. thaliana gene. The indiegogo mentions using A. thaliana... does that get around the laws?

Is there something that after X number of natural generations, the plant seeds are no longer considered GMO?

-Nathan

[Cathal Garvey]

-----BEGIN PGP SIGNED MESSAGE-----
Hash: SHA256

Sorry, didn't follow the link; was only answering the question as I
saw it: "a gene native to many plant species". Which doesn't, by
itself, mean cisgenic.

Yes, if it's from A.thaliana, then you can use it without a license in
most EU states as it doesn't count as "Transgenic".

And no, there's no generational lapse to GMO status in Europe. Once
you put the black magic in, EU law regards it as black magic forever.
However, you can undergo a process of "deregulation" for GMOs, if you
can "prove" that it's totally safe. In practise, doing so is harder
than getting a drug approved, and in most countries it's ruinously
expensive. So, it virtually never happens: the only guys who can
afford to do it are the guys the law was written by idiots to impede:
megacorps.

On 04/24/2013 10:45 PM, Nathan McCorkle wrote:
> Cathal, the paper mentions it IS an A. thaliana gene. The indiegogo
> mentions using A. thaliana... does that get around the laws?
>
> Is there something that after X number of natural generations, the
> plant seeds are no longer considered GMO?
>
>
> On Wed, Apr 24, 2013 at 2:25 PM, Cathal Garvey
> <cathal...@cathalgarvey.me

> <mailto:cathal...@cathalgarvey.me>> wrote:
>
> Nope. "Cisgenic" means "from same species", although some liberal
> interpretations say "same genus". Assume the former. So unless it's
> A.thaliana DNA into another strain/cultivar of A.thaliana, it's
> transgenic.
>
> On 04/24/2013 10:04 PM, Mega wrote:
>> Newsupdate...
>
>> Included this gene into a design which is native to many plant
>> species....
>> http://journals2005.pasteur.ac.ir/NB/23(9)/1177%20-%201180.pdf
>
>> Confers resistance to kanamycin when overexpressed. Does not work
>> in bacteria.
>
>
>> Could you call such a glowing plant "cisgenic" instead of
>> transgenic ? Because it is a native plant gene (and as a marker
>> not GFP but Lux - but both just optical markers, used many
>> times, not toxic)
>
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[Nathan McCorkle]

Can you just start with an already approved GMO strain, and add on mods then without needing to get re-approved? Glowing corn kernels would be pretty damn sweet!

-Nathan

[Nathan McCorkle]

Even if they didn't glow, you could squash them without any sadness like there is when you squash a fire fly. (I did that as a child but looking back it was a mean wasteful thing to do) When the smashed kernel contacted oxygen maybe it would glow?

--
-Nathan

[Mega]

Yes, if it's from A.thaliana, then you can use it without a license in 
most EU states as it doesn't count as "Transgenic". 

And no, there's no generational lapse to GMO status in Europe. Once 
you put the black magic in, EU law regards it as black magic forever. 
However, you can undergo a process of "deregulation" for GMOs, if you 
can "prove" that it's totally safe. In practise, doing so is harder 
than getting a drug approved, and in most countries it's ruinously 
expensive. So, it virtually never happens: the only guys who can 
afford to do it are the guys the law was written by idiots to impede: 
megacorps. 

Didn't know that!

But this would be amazing if possible! 

Can it also be called "cisgenic" with some very common markers (GFP or Lux)? Or is that absolutely impossible?? 

 

[Cathal Garvey (Phone)]

In some countries, apparently including the UK, common marker genes are deregulated (ironically, given antibiotic resistance was one of the scaremongering things used to justify the law in the first place..). However, in others this isn't the case. Not here in Ireland as far as I know.

Also, whether "common markers" are maintained in an easily accessible list somewhere is probably down to a country's implementation.

[Mega]

The kickstarter campaign has finally reached its goal... After just 2.5 days!

Seems there is strong public demand for glowing plantlets... :)

[Mega]

 Just recieved the primers. Wanted to try them out using Phusion proof-reading polymerase... 

However, we switched on the thermocycler, pipetted the liquids into PCR tubes, and wanted to put hem into the thermocycler. Black screen, it died while in stand-by!! The thermocycler is relatively new, 2009/10 and not used every day... The default manual gives  no troubleshooting (just 2 sentences) and a better manual can be ordered for 200 Euros. Whoopie.

Synbio: 1   -   traditional genetic engineering: 0

 

[Koeng]

I thought my thermocycler was terrible....

[Mega]

The PCR didn't yield any fragments (but the ~50 bp Primers). Both from Genomic V.Fischeri (pure culture from Deutsche Sammlung Mikroorganismen)  and pVIB.


So we did some troubleshooting, and noticed that the error is very likely that my professor programed the thermcycler to 95°C instead of the 98°C mentioned many times in the description. At that temperature, the antibody from the Phusion polymerase still inhibits its work, we assume.


At the time, he told me that it's just for denaturing, and 95°C would be absolutely sufficient. Seems not.

[Dakota Hamill]

Was pVIB the control? Did you do an initial denaturation? Was it a
HotStart- Phusion?

I've been having a lot of trouble with primer dimers myself lately but
after a lot of runs and narrowing things down, finally figured out
that it was poor template DNA (or lack there-of) that caused the
problem. I've never worked with Phusion before, but perhaps that 98C
would make the critical difference.

Keep us posted, cool stuff!

[Cathal Garvey]

Yea, 95 should be enough but for antibody-blocked pols you'll need an
"activation" initial denaturation of maybe 5 mins first. Not too long,
or you'll start deaminating the T's in your DNA.

What's the program vs. amplicon length and primer melting temperatures?
How much Magnesium are you adding, if it's not already in the buffer?

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[Mega]

Magnesium was in the puffer, so we didn't change anything.

template LuxCDABEG is 6,3 kbp long.

The genome was rather control, because the plasmid from a recent midiprep  should be purer (did also a proof-of-plasmid-transformation and they glowed).

 Did you do an initial denaturation?

yes, like described in the instruction. https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530

What's the program vs. amplicon length and primer melting temperatures?

It is written 15-30 seconds per kb  , so we chose 180 seconds. 51.4 and 51.5 °C were the primer melting temperatures, and Stefan from the AEC said they always go a bit above the theoretical value to get a better result -> 52°C should work well.

[Mega]

P.S. Seems like a score of  2:0 for synthetic biology  :D

But we knew it would be *some* work ;)  No better waste of time than constructing glowing plant DNA though :D

At the end we'll have the direct comparison between the synthetic construct and the traditional work... In regard of working time, number of attempts, costs (primer+consumables vs. synthesis) et cetera.

[Nathan McCorkle]

On Wed, May 1, 2013 at 7:33 AM, Dakota Hamill <dko...@gmail.com> wrote:
> Was pVIB the control? Did you do an initial denaturation? Was it a
> HotStart- Phusion?

I second this question, where's the control lane? Control in this case
would not be genomic, it would be a plasmid or linear DNA and primers
KNOWN to work for an amplicon of KNOWN size. This way you can
understand whether your polymerase is actually doing anything.

--
-Nathan

[Mega]

Never heared of that before. You mean e.g. doing also a PCR of e.g. a pUC19 fragment in paralell to make sure the polymerase works?

But that will need different annealing temperatures. In a seperate reaction?

I would have said, if there's a 6.3kbp fragment, the PCR was succesful?

[Nathan McCorkle]

Yes that's what I mean. Often a polymerase/PCR kit will come with control DNA and primers. You might need to use different temperature profile, but the point is to test the chemicals and enzymes. So make sure you use all the same water, buffer, nucleotides, etc...

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[Mega]

Ah, ok. Gotta ask wheteher we have one.

Btw.
Seems it was really those 3°C.

[Brian Degger]

pretty much all maize/corn feed will be gm 

----------------------------------------
Brian Degger

twitter: @drbrian

http://makerspace.org.uk

http://transitlab.org

----------------------------------------

[Nathan McCorkle]

Bump. Would that be a legit way to get around GMO approval? (that is, GMing a GMO)

[Cathal Garvey (Phone)]

Not a hope: law in EU is, while more paranoid, certainly better implemented to do what it's intended to than the US. That is, there's no "forbidden" or "permitted" lists (Ahem select agents), instead *Every* GMO is taken as a separate entity requiring a risk assessment.

So, in EU a gmo of a gmo is a new gmo.

[Nathan McCorkle]

would some sort of site-directed mutagenesis count? Select for cells with lots of repeating junk and mutate it into what you want.

[Mega]

Don't know that. Perhapas yes. That's the problem, The american law system is based on how similar cases were decided earlier . If there's no smilar case, if rather may be legal.  In Europe, no chance - GMO = bad.

But what is strange: I heared it's totaly legal to expose plants to radioactivity and then choose the mutants with beneficial traits. That is paranoid, because targeted mutation is forbidden, but random mutatuin with all dangers is allowed? 

Nathan McCorkle <nmz...@gmail.com> wrote:

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--
-Nathan

[Nathan McCorkle]

I'm specifically talking about the EU. I'm not sure there are much in
the way of federal laws banning GMO plants in the U.S.. I know local
laws can make an impact on crops planted, but is it wrong to think
certification isn't required if it's not immediately (a few trophic
levels) going into the human food chain?

> https://groups.google.com/d/msg/diybio/-/17DEEGLOKPQJ.

[Cathal Garvey (Phone)]

Yup. "Mutation breeding" is totally legal, but you'll be breeding for a very long time if bioluminescence is the aim.

[loïc lauréote]

"But what is strange: I heared it's totaly legal to expose plants to radioactivity and then choose the mutants with beneficial traits. That is paranoid, because targeted mutation is forbidden, but random mutatuin with all dangers is allowed? "====> nonsense

2013/5/6 Cathal Garvey (Phone) <cathal...@cathalgarvey.me>

[Brian Degger]

directed evolution is fine, they still need to be registered  

atomic gardens 

http://www.amusingplanet.com/2013/03/atomic-gardening-breeding-plants-with.html

cheers

Brian

[Mega]

I meant targeted mutation in the sense of genetic engineering. When inserting GFP you can be quite sure it won't be poisonous thereafeter. When you expose it to radioactivity, anything can happen, because they're just random mutations.

[loïc lauréote]

spountaneous random mutation happens everyday in the nature, because of sun UV, chemicals products, etc, by biological process.

2013/5/6 Mega <masters...@gmail.com>

I meant targeted mutation in the sense of genetic engineering. When inserting GFP you can be quite sure it won't be poisonous thereafeter. When you expose it to radioactivity, anything can happen, because they're just random mutations.

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[Mega]

You are right. It happens in nature. (by the way, in nature less radiation is applied, so it doesn't mutate that quickly. ) But in my opinion I can't understand that in Europe it is illegal to e.g. insert GFP into plants, while it is allowed to radiate it.

GFP is proven a million times to be ok, and with radiation you simply don't know what the result will be like. Everytime there will be different results, most likely not being toxic. but you just don't know.

[Mega [Andreas Stuermer]]

As for the pGlowroplast I'm having partly synthesized at the moment (yes, they are slowly :D ):

I noticed that the genomic Vibrio fischeri fragment in pVIB (containing the Lux operon) is NOT from Vibrio fischeri ES114 but from starain MJ11. Had to learn it the hard way, restriction with BglII and SalI did not yield 

BglII---RBS-LuxC-RBS-LuxD-RBS-LuxA-RBS-LuxB-RBS-LuxE-RBS-LuxG-terminator-salI
(roughly 6,5 kbp)

But rather
BglII--part of LuxD-RBS-LuxA-RBS-LuxB-RBS-LuxE-RBS-LuxG-terminator-salI
(~ between 4 and 5 kbp)

Fortunately recognized it on a gel before taking it as the fragment.

[Mega [Andreas Stuermer]]

Here's the sequence, attached.

Would have been a lot easier if just synthesized in my vector backbone. But I'm still on a shoestring budget, so a PCR (nowadays) is still significantly cheaper.

[Mega [Andreas Stuermer]]

> The new wave of euroamerican immgration, not sparked by a potato famine or men wearing kilts, but a science freedom famine :P

(or is there a society more *free* than the U.S to do DIYbio/startup-biotech?)

I came to think of one. The island Richard Branson owns? Cayman Island?  Is some billionaire out there wanting to create "free science land" wher all plants are glowing and Neanderthals and dinosaurs are de-extinced? GFP-Neanderthal would also look nice. Just saying^^