Isolating sequences from scratch

[Nico B.]

Hi,

I'm maybe 4 months into any semblance of synbio/molecular bio/bio hacking and have a few projects running because of the amazing support at Berkeley Biolabs and this online community, so for that thanks.

Maybe you've seen pictures of the Diphylleia grayi (skeleton flower) that have been making their way across the internet. I would love to learn to isolate genes -in this instance, that which is responsible for the effect of translucency of the pedals when it comes into contact with water- and have no idea as to how to do this once I've got the physical plant in my possession.

HALP!

Thanks,
Nico

[scoc...@gmail.com]

http://bioinformatics.dnalc.org/gmo/animation/pdf/Detecting%20GM%20Foods%20by%20PCR.pdf

I use this protocol for extracting genomic DNA from plants daily. Its not ultrapure but will last a while. I've had genomic arabidopsis DNA using this protocol for three years and its still useable.

Then design primers to isolate the gene along with restriction digest sites (if you are doing traditional cloning), run a pcr, cut and clean the bands, digest it along with the plasmid, run another gel to purify the sample, ligate it together, transform into bacteria.

If you want to transfer genes u need to find some agrobacterium from a lab and a transformation vector. Then clone in the gene along with a promoter (CaMV or zea mays ubiquitin for example). You should also try your hand at tissue culture and start with a simple organism. Tobacco is my main plant. Don't be fooled by the simplicity of arabisopsis floral dip. Its terribly inefficient and the flowers are tiny and do not last long.  See attached images for tobacco flower. It takes five weeks to get a GMO via tissue culture post agro transformation. I do this stuff all the time. If you want more info, feel free to ask. I love helping people learn more about plant genetic engineering. Best of luck and don't forget to have fun!

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D

---

From: Nico B.
Sent: ‎11/‎23/‎2014 7:03 PM
To: diy...@googlegroups.com
Subject: [DIYbio] Isolating sequences from scratch

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[SC]

>Then design primers to isolate the gene

 

I think when the OP stated he wanted to learn to isolate the gene, what he may have meant was to determine which gene was reponsible for that particular trait.  That's a bit trickier.  I would suggest generating a complete genome sequence followed by some (a lot) of analysis of the data.  Keep in mind that the translucency you are interested in is probably the result of a bunch of genes.  Also keep in mind that plant genome sequencing requires a healthy budget for the libraries/sequencing.  You can start off small though, then continue as resources allow.

 

Neat project, and beautiful plant.

 

 

[Nico B.]

SC, Undoubtedly so.

How would you recommend starting, learning, and working through the process? and what sort of budget are you implying for a project of this size?

I'd love to see this artistically in ornamentals as well as utilizing this as a 'sensor.'

[Dakota Hamill]

Probably tens of thousands.  How deep have you dug into the literature to see if there are any experts on this plant?   You might find crazy botanist who is obsessed with it, though someone may have never published papers on it if it hadn't been used in natural medicine or something of that nature.

To be honest, to me it seems like it's more of a physics question than a biology question, if that even makes sense.  Think about getting white paper wet, it appears translucent because of differences in indexes of refraction etc.     I don't recall all the necessary physics, optics, and waves equations to explain it, but with some reading or another explanation from someone on here more versed in that field, it might make sense.

If you can't find specific genes for this plant which I imagine might be hard since it's not a model organism, you could at least explore another flowering plant with white petals, and learn about the biology of that, including the formation of its petals, pigmentation (or lack there of)

https://www.google.com/search?q=mesophyll&num=40&es_sm=93&source=lnms&tbm=isch&sa=X&ei=NfZzVIa-DOHasAT70oCYCw&ved=0CAgQ_AUoAQ&biw=1242&bih=606

Mesophylls n things?  

It is odd looking...but very pretty.  I wonder if there was an evolutionary reason for it.  There are no doubt tons of papers published on genes encoding petal pigmentation, but I think the ultimate question is:

Is there a chemical reaction going on causing the pigmentation in the petals to vanish, or is the design of the flower in terms of thickness and other physical parameters just right at which point it appears translucent upon getting soaked with rain water?   I'd go with the latter.

"The plant can be found growing on moist, wooded mountainsides in the colder regions of Japan and China come late spring."  Maybe there is a reason for that based on the environment in which it grows?  Maybe there is some special insect drawn to it for some reason more so when the petals are white or clear? 

http://www.efloras.org/florataxon.aspx?flora_id=1&taxon_id=110339

http://www.efloras.org/florataxon.aspx?flora_id=2&taxon_id=110339

It's so cool all those insane words are used to perfectly describe the plant.  One little organism!  Now you got me side tracked reading about flowering plants in the hills of Japan.

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[SC]

Hi Nico,

 

The cheapest way to get going would be to make a large insert shotgun library.  You'll need access to live plant tissue for that; if you don't have access to live plant tissue, you can make a small insert library.  That would be followed by some plasmid preps (not expensive), and some end-sequencing (a little expensive).  

 

I'd be happy to help you through the project. 

 

To start with, do you have access to live tissue?  Is this species wild-collected or cultivated?

 

Stacy

[Mega [Andreas Stuermer]]

I was wondering. In the roots there will be the highest ammount of detox enzymes? Compare the proteome with leaf proteom and see which enzmes are overexpressed?

[Yuriy Fazylov]

Try to find other plants that share this feature.

I would assume that these flower petals are hygroscopic where as most flowering plants are hydrophobic or super hydrophobic.
in addition to having that feature they either drop the pigment or don't have one to begin with. Maybe it has surface features that scatter white light when not wet.
If it has evolved that trait then it must rely on a pollinator that cannot see the flower in wet conditions (assuming the pollinator can only see in vis.). Does it gain optic qualities when wet outside of vis.? How much do you know about the flower?

You wouldn't be interested in fenestration by any chance. Would you?

[Nathan McCorkle]

On Fri, Nov 21, 2014 at 7:26 PM, Nico B.
<perpetuatet...@gmail.com> wrote:
> that which is responsible for the effect of
> translucency of the pedals when it comes into contact with water-

Annotating genes with function usually requires mutants that lack the
quality you are interested in. So you would need a plant that turns
clear when wet, and also a similar plant (ideally the same species and
cultivar) that does not turn clear when wet. Then you should be able
to remove common factors, and the difference points to a function that
is directly related to your trait of interest. If the pathway is
large, you would have to have many non-clarifying mutants that each
possessed a different mutation (and thus would point you to another
part of the trait's dependencies).

This 'subtraction of commonalities' can be done in several ways....
you could get both plants completely sequenced and then find the
difference in software, or you could do a more molecular approach
where you bind all the mRNA to beads, make cDNA, bind the cDNA to
different beads, and use it to 'soak up' the common mRNAs in the
mutant plant, leaving the difference in solution (which you would then
get sequenced). The latter approach is simplified, and would miss
mutations that didn't end up in mRNA (for example mutations in the
promoter regions), but a similar approach might work for DNA using a
FISH approach where your probe was a clarifying-plant DNA fragment
(using every possible fragment of the clarifying-plant genome, aka a
library).

I like the latter idea, since it reduces the computational workload
and instead uses physics as the computer, but it is certainly a
challenging problem and there are lots of ways to go about it... so
you need to weigh your options and hope you choose the path that makes
the most sense for the problem and resources you have available.

[Mega [Andreas Stuermer]]

If the next gen sequencers are as cheap as they claim, you just sequence 10 related plant species and blast for differences. God, I miss that future :D 

[Nico B.]

Hey Stacy,

If I were to order the plant today it would take a few weeks to arrive.

In interim, how could I go about prepping? I'm a novice and am not extensively familiar with these terms and procedures. How would I go about making a large insert shotgun library and the following steps of plasmid preps and end-sequencing? not to mention, what exactly would the end sequencing help me accomplish?

Its easy to order the plants online, seeds as well. however I'm not sure when I will have a blooming flower (tissue), once I receive seeds/plant. Its cultivated.

Thanks,
Nico

[SC]

Hi Nico,

 

I would suggest practicing the techniques on a readily available plant, like a cheap houseplant.   Once you can do library prep easily, then send for the target plant.   

 

Plants have large genomes, so doing the entire genome with the usual combination of PacBio/Illumina assembly would be expensive, in excess of $100K.  Instead, if you make a clone library you can identify the likely genomic fragment by doing a relatively inexpensive end-sequence for each clone and comparing it to the plant species with known genome sequence.  It is more of an exclusionary process:  you can eliminate the clones which are not likely to contribute to the trait.  For example, genes involved in central metabolism will not influence flower morphology.  The clones you are left with are the ones to focus on.  That;s the fun part: you can compare clone sequence to the many mRNA sequences of plants known to be expressed in flowers only (as opposed to leaves, roots, etc.)

 

As always, the more you can do yourself,  the cheaper it will be.  Library construction and plasmid prep can be done in your home or a community lab or school.  Sequencing will probably have to be sent out unless you know someone with a sequencer.

 

I am in th DC area - are you nearby?  I would be happy to show you how to make a library.  I do this very often.

[Patrik D'haeseleer]

I think that trying to address this from a genetics angle is really premature.

I'd start by checking the literature to see if anything at all is known about this phenomenon. Or failing that, if there is anyone who is working on this family of plants that you could contact for information.

Next, I would do some microscopy to figure out what exactly it is about these petal cells that makes them so different from ordinary white petals. Maybe learn how to do oil immersion microscopy, and how to stain different parts of the plant cell.

My first guess would be that this might be a mutation in the biosynthesis pathways for the waxy cuticle that normally coats leaves and petals and makes the surface hydrophobic. Just watching under a dissection microscope how droplets bead up (or don't) on the petals might tell you a lot already. If you know some people who work in a plant bio lab, you could even try to extract the cuticle from a batch of flowers and analyze its composition.

Once you have a better grasp of the physiological mechanism behind this phenomenon, it will be sooo much easier to figure out which genes are involved...

Patrik