Sciencemadness Discussion Board - Trying my hand at genetically modifying plants

Trying my hand at genetically modifying plants

Nate - 22-2-2019 at 11:55

Hey again! So, as a side project other than school I became more and more fascinated with plants. I also love the idea of changing a plant's color through GMO. Literally 5 minutes ago I learnt that hydrangea's can change color depending on soil pH. I was wondering how I could do this, would I use a base plasmid and use gibson assembly to insert the DNA for the dye? I also can't seem to find the gene for the dye, if you could help me look for this that would be great.

Thanks in advanced!

Tsjerk - 22-2-2019 at 13:06

I don't know which one you need to target, but remember there is probably more than one copy.

Nate - 22-2-2019 at 15:13

Yeah, probably right. I need to find out which ones though.

mayko - 22-2-2019 at 16:48

hydrangea pigments are anthocyanins; this looks like a good place to start on their molecular biology (it includes a section on changing petunia colors via genetic engineering).
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC160913/

Metacelsus - 23-2-2019 at 05:26

If you want to make white flowers, using RNA interference would be a relatively easy way to suppress the pigment-producing genes. See for example: https://www.ncbi.nlm.nih.gov/pubmed/18369790

Tsjerk - 23-2-2019 at 06:08

Quote: Originally posted by Metacelsus

If you want to make white flowers, using RNA interference would be a relatively easy way to suppress the pigment-producing genes. See for example: https://www.ncbi.nlm.nih.gov/pubmed/18369790

Probably a good way to go, maybe you can design your RNAi to target a domain specific to your protein

Nate - 23-2-2019 at 21:02

I don't want to make white flowers, I want to take the pigment gene and put it inside of another plant.

[Edited on 24-2-2019 by Nate]

Nate - 23-2-2019 at 21:54

I found the pigment! I have yet to find the gene for it but I have learned the name of the pigment. As stated before it's a pH-dependent pigment, it's not a protein like I thought. The compound's name is delphinidin-3- glucoside. I'll continue to look for the gene in the NCBI database.

Edit: Can someone tell me how to prepare Mutashige and Skoog media?

[Edited on 24-2-2019 by Nate]

Metacelsus - 24-2-2019 at 00:32

https://en.wikipedia.org/wiki/Murashige_and_Skoog_medium#Ing...

You'll need some way to sterilize it; do you have an autoclave?

Nate - 24-2-2019 at 01:57

No, but I do have a pressure cooker. I really need to invest in one.

RogueRose - 24-2-2019 at 17:16

I've been looking into this (on a minimal level b/c I don't fully understand it). What I'm wondering is if you can take a plant that produces some kind of alkaloid or material (the sap, or material in the sap) and engineer another plant (possibly in the same family/genus) to produce the same alkaloid or material.

The ones I was most interested in are rubber latex (so rubber can be produced from something other than the rubber tree) and opium poppies.

For the rubber latex, it would be REALLY great if a plant could be engineered that is grown as a perennial crop, like corn, instead of having to rely on plantations of 7-25yr old trees for production. These plants are a clonal monoculture (all spawns/clones from one source) and are very susceptible to a blight that could destroy an entire nation or continent's rubber trees. If it was an annual crop, this wouldn't be such a big issue if you loose one year's crop and if a disease appeared, destroying the crops is much easier than having to destroy a forest that takes 7 years to reach production stage (and only 15 years of production per tree on average). It would also be great if a plant that could be grown in less tropical climates could be created so we aren't dependent on Asia for 80-90% of rubber output. If the production amount could also be increased, that would be a HUGE help.

If you want to make SERIOUS moeny (like many billions), then work on finding a way to engineer a blight resistant rubber tree. w/o rubber, our world would be F'd b/c synthetic rubber is no where near the same properties. Natural rubber is really an amazing substance - you will find it in some high end electronics (power tools - Matabo, Hilti & maybe some good hot plate stirrers, b/c they are more resistant to higher heat, bending etc, but the process to make the power cords is much more expensive - you have to mold the cord with molten lead that cools & contracts to be removed once it is cooled. This acts as a compression to give it perfect form & part of the vulcanization process I believe).

I'm wondering if plants like poison hemlock, milkweed and others that produce a "latex" like material could be modified to produce a "rubber latex" since they already have similar sap's.

As far as the opium poppy, being able to engineer what alkaloids are produced instead of it producing 7-20 different alkaloids, would be very helpful in the pharmaceutical industry. If another plant (that grow larger in size) could be made to produce these alkaloids in a perennial crop, that would also be a huge benefit. All of this would also reduce the funding in drug running and terrorism $$ that comes from the sale of opium from "those regions", where pharmaceutical companies buy raw opium for production of their products (IDK the %, there are many legit poppy crops like in Turkey, Tasmania and I think France).

Is there a name for trying to get a plant to produce an alkaloid other than what it normally produces? Might it be easier if the alkaloid that it already produces is similar in molecular makeup to the desired compound, or does it really matter if it is being engineered from the ground up.

[Edited on 2-25-2019 by RogueRose]

Metacelsus - 25-2-2019 at 00:19

Quote: Originally posted by RogueRose

As far as the opium poppy, being able to engineer what alkaloids are produced instead of it producing 7-20 different alkaloids, would be very helpful in the pharmaceutical industry. If another plant (that grow larger in size) could be made to produce these alkaloids in a perennial crop, that would also be a huge benefit. All of this would also reduce the funding in drug running and terrorism $$ that comes from the sale of opium from "those regions", where pharmaceutical companies buy raw opium for production of their products (IDK the %, there are many legit poppy crops like in Turkey, Tasmania and I think France).

See this paper from 3 years ago which uses yeast: https://www.ncbi.nlm.nih.gov/pubmed/26272907

I was at a seminar given by Prof. Smolke last year and she mentioned her lab is working on some improvements to the process. (Also see here: http://smolkelab.weebly.com/research.html)

karlos³ - 2-3-2019 at 22:54

Quote: Originally posted by RogueRose

Is there a name for trying to get a plant to produce an alkaloid other than what it normally produces? Might it be easier if the alkaloid that it already produces is similar in molecular makeup to the desired compound, or does it really matter if it is being engineered from the ground up.

[Edited on 2-25-2019 by RogueRose]

This is simply called biotechnology

It involves all the processes being used to enslave some organism to produce an organic chemical, be it a pharmaceutical, an intermediate, even the precursors to pharmaceuticals.

It depends on the organism of course, also the ability can be engineerded into an organism, but some produce already unengineered some precursors which is unrelated to compounds produced by them, usually if something unrelated to it is added to a culture of them.
For example amino alcohols being made from a precursor produced by yeast(famous for l-PAC, less famously for example (S)-3-chlorophenylpropanol from the corresponding ketone).

DrRadium - 7-3-2022 at 11:28

Quote: Originally posted by Nate

No, but I do have a pressure cooker.

You have an autoclave. Autoclaves are just specialized pressure cookers. You put some water in the bottom of the pressure cooker. Put all the components of the desired media plus agar mixed with water in a suitable bottle or erlenmeyer flask, cover top with aluminum foil or loose cap. Put top on pressure cooker, heat up until water boiling and steam blowing out vent. Put the weight on the vent or activate whatever the mechanism is for holding the steam in under pressure. Heat to 15psi, hold there for 20 minutes, shut off heat, let cool until pressure back at atmospheric and below boiling. Cool to about 65-70°C. This is cool enough you can just barely keep your hand on the flask indefinitely. Take out flask/bottle, swirl, add any antibiotics or other heat sensitive components needed for plates. These should be filter sterilized through a syringe filter or similar filter for sterilization. swirl to mix, then pour as any of the many video examples on you tube such as this one:
https://www.youtube.com/watch?v=ey19jM6y7-c

That's it for autoclaving and pouring agar plates.

Chalo - 27-8-2022 at 17:55

Don't be discouraged, but it is rare to have a single gene make a pigment. For example, human melanin production requires at least eight genes. You should also be aware that many plants are poyploid (have more than a pair of each chromosome). For example, potatos are tetraploid which makes the result of changing one gene unpredictable.
I am ettaching an excelent general guide to plant biotechnology for you.

Chalo - 27-8-2022 at 17:56

OOps, messed up the attachment but here it is now

Attachment: agrobook 03 plant biotech.pdf (441kB)
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Mateo_swe - 21-2-2023 at 06:34

If you want some info about DIY sterile teqniques look at the mushroom growing comunity, they got all info about pressure cooker sterilizations, glove boxes, sterile flow hoods etc.
There are also many pdf ebooks about sterile plant growing, modifying plants and similar such things.
I attach a PDF about tissue culture growing.

Attachment: Plants_From_Test_Tubes_Complete.pdf (3.6MB)
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Attachment: Tissue culture of ornamental cacti.pdf (1.9MB)
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DrRadium - 29-12-2023 at 02:20

Quote: Originally posted by Metacelsus

Quote: Originally posted by RogueRose

Let me dust off my copy of the Necronomicon and get the candles and my black robe out. Threads only 4 years old, only MOSTLY dead really, hardly even a necropost....

A more accessible target for psychopharmaco-biotechnology is the pathway making psilocybin. Only 4 genes, all cloned, with proof that simple co-expression of all 4 results in biosynthesis of the desired product.
https://pubmed.ncbi.nlm.nih.gov/28763571/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232020/

This makes possible something that absolutely no one should actually do without appropriate licensure by pertinent authorities but with which I came up with as a thought experiment. In the early 1990's while drinking homebrew with other grad students, we were talking about prodrugs and musing over 'hallucinogen-ogens' (or a preprodrug and thus a hallucinogen-ogen-ogen). I cooked up a way to use yeast biology to produce 2 transgenic yeast strains that only produce psilocybin when mixed together... a hallucinogen-ogenic yeast. When apart, they don't make anything subjectively psychoactive except alcohol.

You just use standard lab haploid strains, one that is constitutively "a" in mating type and another that is "alpha' mating type. Half the pathway in one strain, half in the other. Since there are 4 gene products in the pathway, Put two genes in one strain, two in the other, with each gene under its own promoter/enhancer and with its own polyA site. Put the two transcriptional units one after the other as a cassette that insertionally inactivates the URA3 gene on a cloning vector with the chromosmal URA3 locus and flanking DNA needed for recombination. These are commercially available. Transform the yeast with the linearized construct and select with 5-FOA in minimal media plates for ura3- colonies. Most will be recombinants with the disrupted URA3 gene expressing the two psilocybin synthesis genes. Inserting the transgenes in this way is vastly more stable in the absence of selection pressure than any type of plasmid or even an artificial chromosome. Especially in haploids where there is no functional copy of URA3 on a second chromosome 5 with which to recombine. Recombinants are the way to go when stable yeast transgenics are grown on rich media. Just as everyone uses URA3 because negative selection with 5-FOA is so easy. Thus the components being commercially available.

Mating type "a" strain will express psiH (tryp-4-monooxygenase, usual substrate tryptamine, product 4-OH tryptamine) and psiM (transmethylase, usual substrate phospho-4-OH-tryptamine, product is psilocybin). Mating type 'alpha' strain will express psiD (decarboxylase, usual substrate tryptophan, product tryptamine), and psiK (4-OH tryp-kinase, usual substrate 4-OH tryptamine, product phospho-4-OH-tryptamine). They grow fine that way and each strain will not produce psilocin, psilocybin, or baeocystin. 4-OH tryptamine isn't psychedelic (it will probably be produced in strain "a" due to endogenous aromatic amino acid decarboxylases).

But mix the two strains together and they will immediately mate to form diploids. Diploids grow faster than the haploids, and will rapidly take over the culture; They also possess the entire psilocybin biosynthetic pathway. Diploids will produce psilocybin, and thus will make psychedelic beer, wine, and bread. The diploid strain can be propagated indefinitely on rich media and will only sporulate under starvation. Though it may start to produce less psilocybin as is continually cultured since recombination may start to inactivate psi genes in some some cells.

There are ways to increase yield, but other expression systems are probably better for trying to maximize raw yield. As I said, a this was a thought experiment, it only became something possible to do in 2017 when the genes in the pathway were cloned.

Please do not try this at home, or in a lab, or anyplace else without licenses and approval from whatever your local version of The Man happens to be. If you do, remember I told you not to. Even if legal where you are and prison isn't a concern for you, this yeast will look like any other yeast and someone consuming it accidentally could be harmed, at least psychologically. I can't and won't provide any more details about how to do this, at least to anyone who doesn't have the necessary DEA (or your local equivalent) license to do so. I know....I'm a killjoy. I tell you it's possible how to make psilo-beer and bread then tell you not to do it. What can I say, I'm old and like keeping the government licenses that allow me to make a living.

OTOH, I swear that if a single gene or similar simple method for introducing high level spider mite resistance in edible plants is published, I will make my own bioengineered french fillet green bean cultivar. I am that sick of spider mites destroying my f-ing bean plants every year. If only there was a version of BT parasporal crystal protein that killed them, I'd already be on it.