Monthly Archives: November 2015

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The Little Weed That Could – Intro

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One day, as I was watering the myriad plants I have by my ultra-long window sill, I noticed a funny little weed growing up through the gravel of my lemon tree. It looked like a clover, was soft to the touch, and grew in clumps. Around the lemon pot I also noticed a ton of brown specs. They were all over the window sill, the floor, and even on some of the leaves of the plants just below the citrus. A deep and sudden chill shot through my spine as I feared the worst – insect infestation! Being more curious than cautious, I picked up one of the little critters off the floor and immediately notices some lateral ridges across the width of the thing. It was burnt orange in color, tear shaped, and look very scale-like. I tried to crunch it between my nails with no avail and thought it too thought to be an insect or egg. I followed the trail of specs to where they were most concentrated and oddly enough the central mass surrounded the little clover-like weed. Luckily the weeds were in bloom and one of the bracts had set seed. It looked like a tiny okra and one was burst wide open. A single brown spec similar to the ones on the floor clung to the seed pod for dear life. A SEED! I thought, as I wave of relief rushed over me. It was just the seed from the weed-let growing in the citrus pot. Immediately, a curious thought came to mind: How does such a small plant produce such a vast amount of seeds? Has it really been here for THAT long that I failed to notice it nor its insect-like spawn floating around? A bunch of more questions whizzed by as I fiddled with the perfectly symmetrical clover in one hand and barbarically typed in words relating to the plant with the other. After a few minutes I stumbled upon some pictures of the little guy. Oxalis pes-caprae, or so I thought! Its listed as an invasive species native to South Africa and is a fast growing bulb-producing weed that is really hard to get rid of and harder to kill. Link here: Oxalis pes-caprae The leaves are soft and tender to the touch. No sign of waxy cuticle. Each leaf is identical in weight and size (once full expanded) and seeds seem to form as soon as the plant has about 5 or so bracts to its name. More data needs to be generated on growth habits in-vitro and in-situ to determine growth rate, seed to seed time, etc but just judging by the sheer volume of seeds on the floor with little outgrowth I’m fairly certain its fast as hell.

 

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I’ve been hunting for a good plant to use as a test platform for my genetic engineering experiments for some time now. Arabidopsis is cute but its so fragile to the elements that it might as well need a space suit just to live on earth. For the DIY plant biologist, it simply is not the best model for the job no matter how many people swear by it. I’ve seen growth chambers go for $18,000 so it can deliver 100 microeinsteins of light evenly across the entire surface of the shelf. That’s all fine and dandy but when all you have are some grow racks and cool white fluorescent tubes from WalMart, temperature humidity and light control will be tricky to say the least! Also from what I’ve experienced, there is no decent way of standardizing the tissue source used for plant transformation to account for positional effects (petiole to leaf tip varies in efficiency) and overall tissue uniformity due to ridges, texture, or other physical structures on the leaf itself.  There are some personal requirements I have that need to be met in order for me to feel comfortable working with the organism in question. One of the most important is “Sebastian Tolerant”. By this I simply mean the ability to survive neglect. Despite what people may think, I have a terrible green thumb. If it weren’t for my mom, I would probably be in a different field of study due to mortality rate alone! …but I digress.

 

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A hot topic for me these days is visual marker expression. There are a few markers on the market that can be used to tag proteins so you can trace their trafficking or expression rates. Some require special equipment like a fluorescent microscope (easily DIY’d but too much effort) and some require special chemicals like X-Gluc for the GUS assay which can be pricey unless you got the hook-up ;P (more on that later). One attractive possibility is anthocyanins since they are stable and a chemical rather than a protein but more on that in a later post! My most recent interest is in the GUS assay since, despite its cost draw-backs, produces a solid bright blue precipitate at the location of the cleavage enzyme so it wont chemically leach out of the tissue. The entire assay uses a chemical, namely X-Gluc which is colorless and becomes cleaved by the reporter enzyme GUS into said cerulean solid. The catch with this assay is the color itself. While blue is far less common of a color to be found in plants, especially in the leaves roots and stems, it will compete with the dark green of the chlorophyll so slight signals or low concentrations are hard to visualize. The solution is to bleach the tissue with 95%+ Ethanol. It essentially “ghosts” or vitrifies the tissue to a nice white to off-white color while preserving the structure of the plant and more importantly the reacted GUS product therein. The ethanol rapidly dehydrates the cells and the chlorophyll leaches out through the leaf tissue and stomates. A fun project to do if you have some high proof alcohol lying around (think Absinth)! Just shake slow overnight or if you don’t have a shaker just let it sit submerged overnight and give it a swirl every few hours if you have time. Anywho, GUS is tricky since it can lead to false localization signals if the X-Gluc did not penetrate deep enough. The thicker the tissue, the harder it is to get the substrate all up in there. The Oxalis leaves, on the other hand, are thin, flat, and uniform with little to no features across the entire leaf. This makes the tissue ideal for GUS assay for protein accumulation (possible mini-article for 2016?!?).

 

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The second reason why I am excited about the boring texture of the leaves is that it will make for a great alternative to onion skin for my new gene gun! Its been sitting pretty on my lab bench until I have time and funds to get some helium and tungsten particles…hopefully soon! The gene gun, which I will most definitely write more extensively on in the coming months, essential is a nanoparticle shotgun that looks like an esspresso machine. You bind whatever plasmid you want to some ~1um tungsten (gold is the standard but I have moral and financial issues with shooting gold down the drain) nanoparticles using ethanol and spermadine (smells terrible) and then adhere it to a piece of circular kapton tape (YES IT IS KAPTON AN NO ITS NOT SOME FANCY PRESSURE SENSITIVE PLASTIC OF EXOTIC ORIGIN AND MAKEUP). The gene gun the pressurizes and depending on the thickness of the kapton “rupture disk”, it rips open a hole into the disk and the compressed helium escapes. As it expands, it accelerates the disk holding the tungsten particles at incredible speeds under a vacuum (little friction if any) and the disk goes hurtling toward a mesh a small distance away. The steel mesh catches the rupture disk but the holes in the mesh allow the particles to pass through mostly unhindered at the velocity accelerated by the disk. That is the business end of the gene gun. Below the muzzle is a petri dish with neatly arranged sterile plant tissue focused on the center of the dish. The shotgun effect obliterates anything in the dead center but the spread is slow enough to only penetrate and not mushify the outer edge or “corona” of the tissue group. This is the part you hope to have been penetrated by the tungsten. Now if the DNA bound tightly to the tungsten, and the tungsten penetrated the cell wall and cell membrane but did not cause exit wounds, and the DNA wasn’t shred to confetti, a very small percentage of this event will be fit for an integration event. Now, DNA is soluble in water and all life is aquatic so as soon as the tungsten gets inside the cell, it releases the DNA. That is where the magic happens! I’ll leave you in suspense as to how exactly it gets integrated along with a thorough rant about how the leading theories about integration and uptake are, in my opinion, total bullshit. I promise to touch back on the fun and controversial topic at a later date but for now lets get back to the topic at hand – Oxalis.

 

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Due to the uniformity of the leaves, Oxalis pes-caprae would make for a nice leaf transformation platform for gene gun optimization or at least a benchmark of efficiency but none of that would work without a good regeneration and tissue culture protocol. Aside from sowing the seeds and monitoring them like a middle school science fair project, I will start trying to get them to regenerate in-vitro too. The core of any plant genetic engineering project is tissue culture and I cannot stress enough the importance of solid foundational understanding of culture techniques, aseptic work, and an overall instinctive feel for the state of the plant in question. All of these come with practice and I have heard time and time again of newbies to the dark-green arts wanting to transform plant X with gene Y so they can save the world and contribute to science. This is all well and good and I encourage anyone and everyone to try there hand at plant tissue culture but the first thing I always ask is have you every regenerated a plant before. This task is trivial for me now but it sure as hell was NOT trivial for the first few years I struggled learning it by myself. I was almost about to change my field of choice to mycology because I was the king at growing all kinds of beautiful and wonderful fungi…but the intention was to clone basil for my mom’s cooking. I was so disillusioned with tissue culture and everything kept dying left and right that I really wanted to quit. I took a LOOONG hiatus from lab work and plants in general to get over the depressing ordeal of months and months of failure until one day I began to know my enemy. I started looking up the fungal bastards that stole my joy away like Liam Neeson in Taken. I learned all about fungicides and fungal control in-vitro and hashed together a toolkit along with some much needed controlled experiments to finally thwart the invaders. After years I finally got control over what was growing on my plate…and found a deep appreciation for seeds. More on that later. (I really need to write more…too many promises and too little words put to paper…DIGRESSION!)

 

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Anyway I need to start some controlled experiments to get a good regeneration protocol down before I decide to infect it with agrobacterium only to have it die in-vitro due to poor media or culturing protocol. Hormones at the wrong time in the wrong amount can permanently screw up the cell cycles of the tissue and keep it in the dreaded dormant callus state forever. I’m going to start off with my goto media of MS with Gamborg B5 vitamins with 1mg/L BAP (6-benzylaminopurine) and 0.1mg/L NAA (alpha-napthaleneacetic acid) with 100mg/L Timentin (generic fish antibiotic brand of Amoxicillin and Clavulanate). I’ll start some seeds in-vitro with my standard ethanol evaporation surface sterilization since the seeds are small enough to possibly save me some time and limit exposure to bleach. Once the seeds germinate I will wait for the cotyledons to form and then take them along with the hypocotyl (stem from ground to cotyledons) and culture them first. Ill let some grow further and try my hand and regeneration from mature expanded leaves too. Basically chop the whole damn plant into small pieces and plate them all on the same media on the same dish with some spacing. I like to spiral the tissue so I know what region it came from. After that its changing diapers every two weeks and once shoots form, move to rooting media of just MS, Timentin, and NAA at 0.1mg/L until it roots. If that doesn’t work Ill go for 2,4-D and 0.5-2mg/L, Zeatin, etc etc until I find the best mix. I should also hit up some articles dealing with oxalis regeneration or related species (take all protocols for plant tissue culture with grain of salt as they are seldom reproducible 1 to 1 and will take time and tweaking).

 

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Next I’ll do some recon genotyping to ensure A). it acutally IS Oxalis pes-caprae (have a feeling its the creeping wood sorrel not the common yellow wood sorrel). And B). The DNA extraction process is simple enough to use Edwards Buffer or a simple ethanol precipitation. In science, cheap is king! I was thinking of targeting both the RuBisCo large and small subunit (chloroplast and nucleus respectively) as a standard plant barcode and sequence the resulting PCR. I am a little more partial to the chloroplast genome of it since its smaller to fully sequence and the target of all my latest research but a good understanding of the plant is in order if it ever wants to become a shining star alongside Arabidopsis. That most likely will NOT happen but it would be a great learning experience to learn the crap out of a single plant from beginning to end. It may even shine some light on weediness and why its so damn successful as a species. So I’ll PCR the rbcL, rbcS, matK, a few herbicide tolerance genes just for good measure and hopefully a negative control (last thing we want is herbicide resistant bermuda buttercup) along with some homologous integration sites within the chloroplast genome. Last but not least I want to home in on the oxalic acid pathway as my first foray (beginning to end) into the wonderfully inefficient world of CRISPR. I have some ideas for a middleschool/highschool curriculum based on oxalic acid assays (YES I KNOW ITS BEEN DONE BEFORE) and would love to make some knock-outs. It would be such an easy way to determine if its off or on, just mush a leaf in water and mix it 50-50 with high concentrated calcium chloride and if a white precipitate forms, its still producing oxalic acid. So easy a 6 year old can do it…and that’s the whole point! Get those little tykes involved with science early and see what wonders they can accomplish later on in life. Its always all for the kids anyway. Just wish I had cool things growing up… ::sigh::… DIGRESSION!!!

 

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The last thing I will mention but the first thing I will do is the development of an ethical selection marker. I know that sounds silly…plant ethics…but hear me out. All of the transgenic plants, with some exceptions, produce an extra protein on top of the gene being expressed as a means of culling the tissue from the non-transgenic wild type. It normally comes in the form on an enzyme that breaks apart an herbicide molecule and renders it inert…or so they say…DUN DUN DUNNNN 😛 …. This attribute is great for in-vitro work since it kills the non-transgenic tissue and only leaves the ones that are expressing the gene…and by transitive property (but not all the time) your gene of interest as well! When it comes to release, although being that I am NOT IN ANY WAY CONDONING NOR PARTAKING IN THE RELEASE OF ANY TRANSGENIC ORGANISMS WITHOUT PROPER APPROVAL FROM THE POWERS THAT BE, and the fact that Oxalis pes-caprae IS A NOXIOUS WEED AND AN INVASIVE SPECIES, I wont ever let any of these seeds, plants, or soil leave the lab without proper containment, permits, and approval. That being said, having a nuclear-expressing herbicide gene means its will be present in the pollen meaning it can spread around meaning super-weeds meaning end of the wor… No, really its just irresponsible to produce herbicide tolerant plants alongside your gene of interest so there must be an alternative…and there is! Enter Syngenta!

 

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Syngenta, a ag biotech juggernaut, has a nifty little piece of technology that just went off-patent called “positive selection”. This uses a gene from good ole’ E. Coli to give the plants the ability to eat and metabolize a sugar that it normally can’t, namely D-Mannose. Its a bit more expensive than table sugar…okay…like an order of magnitude more expensive, but using this as a means of selection via wild-type starvation ensures that the only thing that makes the GMO plant GMO is your gene of interest (which is its own can of worms) and the ability to eat a sugar that is not so common in nature and wont make mutant super-weeds that will kill us all. All joking aside the rise in herbicide tolerant weeds is becoming a real issue and the science simply cant keep up with Nature’s ability to adapt. There are ways to remove the selection marker post-transformation but its a bit involved and requires some extra genetic parts. What better than a freedom from the use of antibiotics/herbicides in routine transgenics! So I managed to isolate the sequence from my E. Coli strain HB101 (as per the patent) and cloned it into my open source work-horse plasmid p2KB. The next steps will be to add the nopaline synthase promoter to express the protein (phosphomannose isomerase) and the nopaline synthase terminator to end the genetic circuit in good grammar. I heard tales of a terminator from Arabidopsis, namely the 3′ UTR of the Heat Shock Protein 18.2 that increases expression by 17% vs Tnos (Matsui, Sawada, and Kato 2014) but only time will tell.


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Also the way to determine if your plant is transgenic or not is colorful and interesting to boot! The metabolism of sugar acidifies media so using a color indicator like Chlorophenol Red in the media and a hole-punched leaf disk in a small 24 well plate means you can visually inspect for phosphomannose isomerase activity by way of color change in the wells from red to yellow. Of course PCR is the way to go for the final say but the fact you can do it visually makes it all the more enticing!

 

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In short I have a lot to do and a looong way to go but luckily all of these tasks overlap with my zillion other projects and an upcoming publication in PLoS on non-plant related stuff. Yeah, I know…first paper publication will have little to do with plants. Such is life! I promise it will be interesting and shed some light on a dead field of research and the concepts of stress on the molecular level. Yay, science!