Monday, March 31, 2014

Protoplasts!

One of the procedures I am learning is how to generate and transform protoplasts. Protoplasts are really cool. They are single plant cells that have had the cell wall removed but the membrane stays in tact so the cells are still alive. The cell wall of plants are mostly cellulose and resistant to environmental changes to protect the cells. Since cell walls are hard to penetrate it makes it difficult to add any DNA I want into the cells with the cell wall in tact. Which means I have to get the cells away from the cell wall and turn them into protoplasts. So how do we get them out?

 First we use the tape-sandwich method by Wu et al 2009. I put Fisher tape on the top of the leaves and then regular old Scotch tape on the bottom, press the Scotch tape on carefully, then rip that puppy off like I'm waxing eyebrows! It's soo much fun :) As you can see in the above image, the Scotch tape removes the bottom epidermal layer of cells which are very waxy and have to be removed to expose the more sensitive cell walls. The newly exposed leaves are put into a special solution full of enzymes that break down cellulose to remove the cell wall that connects the individual cells. They will fall out into the enzyme solution and I can see them under a microscope.


As you can see in the above picture, there are pretty round protoplast cells.. but there is also a lot debris. Without their cell walls, the protoplast are very sensitive to environmental conditions and shaking.. even pipetting can cause them to burst. They have to be handled extremely carefully and even with great care I still get some loss. Once they are isolated though, I can use chemicals to weaken the plasma membrane slightly to allow the introduction of plasmid DNA to the cells. Then leave them overnight so they can grow and make the gene product that I want. In this learning phase, I'm using simple reporter proteins GFP/GUS.

GFP = Green fluorescent protein.. aka glows green! Under a special microscope I can visualize the glowing protoplasts! It takes 2 days of work, but it is a really cool result! And when we are talking about genetic modification, 2 days is barely a blink of an eye compared to stable transformations. This method will let me test a lot of different genes in a relatively quick fashion to see what works and what does not.

Not to mention, I'm making GLOWING CELLS.. how cool is that?!?! :-D

Friday, March 28, 2014

Quiet House

Boo has been gone all week. It is awful. Parenting is hard, parenting Boo is REALLY hard... and yet when he is gone life is even worse! Yes, I get to work longer in the lab and I do try to take advantage of this. Yes, I could go out without trying to find a baby sitter (which is my least favorite thing ever) but I don't. There are lots of reasons for this. 1) I don't have time when Boo is home to invest in outside of lab relationships. 2) I am not a good loner, I don't go out to eat alone or to movies, etc.

It has been a really long and lonely week with a great deal of time to think. I had planned, of course, to read and get all caught up and surpass my goals for quals. But I come home and I end up sitting on the couch and just staring at the wall. I finally realized that this is the first time since Boo has been born that I have been 100% alone while he is away. Before we moved here for the PhD we lived with my parents so when he would leave I had family to keep me company and keep things hopping.

It's amazing how sad and unable I have been to find my motivation this week. I have bouts of sadness, mommy blues, stressful times, and just plain not a clue why I'm crying into my Pepsi One even when Boo is home. But this week, completely alone, it has been a nightly occurrence.

He comes back on Sunday. It cannot get here fast enough!

Wednesday, March 26, 2014

Plasmid Prep

This week I have been working on generating plasmids in trusty Escherichia coli, aka E. coli. A plasmid is a small circular, replicating piece of DNA. For research, we can put any number of genes in the plasmid and whatever we transform will then make them! Right now I am simply learning the method so my plasmid is generic pCambia 1304, yawn. The nice thing about this plasmid is it has two reporter genes (GFP & GUS) that are on all the time in the cell. So any cell expressing the plasmid will either glow green under UV light (GFP) or fluoresce in a special assay (GUS) making them easy to identify. 

Monday, I put the plasmid into the E. coli cells, it was really easy as we were using special E. coli that is ready to accept new DNA.Then I had to let them be fruitful and multiply.. in a very smelly media. Seriously the media smells like warm trash.. then you let E. coli grow in it for a while and it REALLY smells. Today was a smelly, smelly day! BUT I had a lot of E. coli that would all have copies of my plasmid inside of them... next step, get those suckers out!

That is what I spent all day working on, and I'm still not done! Thankfully the procedure has a few "holding pattern" points where it can run for X minutes to overnight. I stopped at the last holding spot and was the last one out of the lab. Which is weird for me as I'm usually the first to leave every day to get Boo from daycare. But this week he is with his Dad (more on Friday about that), so I could stay late.

Isolating plasmids is a lot more complicated than you might think. Getting DNA is easy, I can literally do it with dish soap, salt water, Everclear, and a strawberry in my kitchen. Plasmids though, they are a different story. Inside every E. coli cell is the cells natural DNA, the long strand of genomic DNA which is 4.6 million base pairs long. I then add a plasmid that is 6128 base pairs long. That is a lot of DNA floating inside this tiny E. coli cell.. and I only want the plasmid! The genomic DNA is actually a big pain in my booty. Thankfully, despite the fact that they are made of the same material, there is a way to separate them.. any guesses about what we use to do that?

Size. Remember genomic is 4.6 MILLION base pairs.. that's a LOT bigger than my plasmid. Perrrrfect! Using size to your advantage is great, but it takes a lot more time and care than your standard crack the cells and rip the DNA out extraction method. If you break that large genomic DNA strand into little ones it will end up in the fraction that you think is all plasmids. The bacteria must be carefully open and all the other macromolecules (fats, carbs, DNA, RNA, etc.) removed with a series of chemical and centrifugation steps.

For this procedure, the centrifuge is the key. Different speeds will isolate out different sized particles. A nice "slow" speed will get all the big stuff, like unbroken E. coli, large chunks of membranes etc. A fast spin will bring down the smaller molecules. By doing a bunch of purification and centrifugation steps, you can slowly get rid of the big stuff and end up with just the little plasmids. Tonight, while I sleep my little plasmids should be precipitating out in their lovely alcohol bath in the freezer. Tomorrow I will wash them and quantify them, hopefully there will be a LOT cause I need a ton for step #2.. transform some Arabidopsis protoplasts :) Hopefully in a future post I'll have pics of some beautiful glowing protoplasts that I made.

Monday, March 24, 2014

Spring is Coming!

At least that is the rumor.. so far I have seen signs of spring but with the snow forecast for tomorrow the stupid groundhog is being proved correct. One of the best parts of spring are the flowers! The white, snowy landscape (or in a city the gray/blackish disgusting slush) gives way to brightly colored flowers of all shapes and sizes! The trees were awake at Boos school this last week, little red buds glittering like rubies.. spring is coming :)

Now to link this to all the tags in this post.. My course this semester is plant development seminar. Seminar courses are usually student led, students take turns presenting a current journal article on the topic. Mine is coming up in 2 weeks and the topic is.. FLOWERING! :-D

The study I have chosen to present is Phloem long-distance delivery of FLOWERING LOCUS T (FT) to the apex by Yoo, et al. The Plant Journal (2013) 75:456-468. Which I apologize is not open access so you can only read the abstract unless you have a subscription to the journal. This article is REALLY cool and full of a ton of things I knew nothing about before I started reading it!

Takehome message of the paper: FT/FTL2 are produced in the companion cells and transferred into sieve elements where it is transported to the shoot apical meristem and initiates flowering.

Say what?! Ok.... ummm.. let us try to break all of that down into normal people speak.

First those FT/FTL2 thingies are simply acronyms for proteins called FLOWERING LOCUS T which has been shown to be the signal that changes the shoot from vegetative (leaves/growing) into reproductive, aka FLOWERS! :)

Next, plants have a vascular system that consists of phloem and xylem. Phloem is for nutrients and it has several different types of cells. The two important for this paper are companion cells and sieve elements. Companion cells have all of the organelles and cell machinery required for making proteins, like the FT. Sieve elements are conduits, they move things around.

Lastly, meristems are stem cells of the plant world. They create new cells that can become any type of plant cell. Meristems are where plants grow.

We can put these all together to say that leaves sense the environment, in this case how long the sun is up. When the right amount of light is perceived, the companion cells start making FT protein. FT protein moves into the sieve elements via diffusion and travels up to the above ground growing point(s), the shoot apical meristem. This meristem shifts from normal growing conditions to flowering conditions and POOF a flower appears :)

That's all I have figured out so far.. good thing I have almost 2 weeks to finish figuring this out! :-D

Thursday, March 20, 2014

It's a Jungle Up There!

First off let me apologize for this post being a day late. I was mixing this video last night when I fell asleep.. :-D

When you leave an easily amused graduate student alone with a mass of tangled, gigantic soybeans, her mind tends to wander to random places. When I pull back the curtain to my soybeans homeland, all I could think was.. wow.. it's a jungle up here! Which got me humming the Monk theme song, come on you all know it! If not:


Being easily amused, I started messing around with the lyrics and my cell phone. This is the result, I hope it makes you giggle, it certainly made me laugh:


Monday, March 17, 2014

First Professional Meeting Talk!

This Saturday, after much panic and stress, I successfully made my first oral presentation at a professional meeting! I have done presentations before, during my MS I gave invitational department seminars and, of course, my defense. But until this point whenever I went to professional meetings, I always had a poster presentation, not a talk! Granted this was a smaller, broad all science in the state meeting and not the international scale I used to attend, but I got to SPEAK! Give my own 12 minute talk.. on a project that is barely getting started. Hence the panic and stress.

As anyone following the blog knows, my research is going really slowly because my soybeans grow really slowly. The idea of standing up in front of people with the little bit of story I have so far was terrifying to me. But by pulling out some of the previous, nonpublished work that led to the creation of our soybeans I was able to put together a pretty nice story.

The conference ran from 8 - 6, with oral presentations from 9:15 - 12, 3:15-4:20. My talk was slated for 4pm, the very last time slot for the sessions. This is not the ideal time slot, where the morning sessions are usually fairly well attended, the later sessions are not. Of course this can be an advantage because the later sessions are not well attended ;)

I worked all week on getting slides together, scripting, practicing, editing... it was almost all consuming. I presented to the lab twice, got lots of great feedback that resulted in even MORE edits. Friday night, I was making new figures/graphics to add to the presentation and tweaking the text yet again. I printed out tiny slides with notes and managed to get all 18 slides onto front/back single sheet of paper that I could fold into my purse to review on Saturday.

Around 3 on Saturday I found a nice bench to lay on in the hotel and read over/practice my notes again. At this point I found a way to improve my slides and talk once again. So I made my way to the presentation room, right before the session started, so I could make an edit an HOUR before my talk! My editing/polishing side needs to simmer down :)

I could not focus on the talk before mine, I was lost in my notes trying to make sure I remembered everything I was sure I would forget. My stomach was rolling in a sea of bile and I altered back and forth between cramps and the desire to vomit. After the lady before me finished taking questions, I walked up, pressing my hands to my legs to keep them from shaking, loaded my presentation and took a deep breath. Then launched into my pre-planned opening statements. As always, the initial adrenaline spike of looking an audience in the face was enough to overpower my nerves and we bounced energetically forward. I hardly looked back at my lab, the sight of my advisor made me too nervous, but I did let my eyes meet with everyone else in the room. There were a few really nice smiles and attentive looks that helped keep my nerves from coming back. I got chuckles and smirks when I was aiming for them and questions that showed people were really paying attention and absorbing what I had to say. Some good feedback at the end from both the lab and strangers in the audience. Overall it was a very successful first talk!

Friday, March 14, 2014

#PositiveParentMoment

One of the things that I have been told repeatedly is that thoughts are like gardens, the ones we tend the most will grow the largest. As anyone can tell you, being a parent is both rewarding and exhausting. I find myself getting overwhelmed with parenting, feels like more often than not. Then after a bit of gentle nudging from my Mom after a particularly bad time, I realized I was feeding those thoughts more often than not.


So at the beginning of the year,  I decided to challenge myself to pay attention to moments that were POSITIVE every day. I created a Twitter handle (@PositiveParent365) with the sole purpose of capturing these moments where I could look back and read them. I had hoped to do them daily (hence the 365), but life gets in the way and I have missed a day here or there. To mark them I've been using #PositiveParentMoment. So far there have been 74 days in the year and I have marked 64 positive entries. I go back and read them occasionally, especially in moments where I am feeling overwhelmed, and it's been working.  Feeding my brain positive thoughts, particularly in potentially non-positive moments, is slowly starting to help me feel better about parenting. My brain has a way to say: Look it's not all a giant waste, some moments are epically awesome, even the little ones. For example:






Three months into this journey and I can see a difference. I still fall back on my old frustrated, overwhelmed ways sometimes but change takes time. But since I started looking for positive things to focus on and memorialize, those times are lessening.

I would love to see more #PositiveParentMoment, if you have Twitter, post your moment! If you don't post one in the comments here! What little moments can we use to feed the garden today?

Wednesday, March 12, 2014

Mammal March Madness Part Deux

Mammal March Madness is in full swing! If you are not following #2014MMM on Twitter, you are missing out on awesomeness. Boo did complete his bracket before the festivities started, but unfortunately had to go to bed before the end of Social Mammals first round but when we went over it after school today and he was delighted. There were lots of WHAT?! REALLY?! WOW! at the amazing facts. To see the first half of his bracket, see the first Mammal March Madness post. Now onto the rest of his picks!

His The Who in the What Now?:
http://mammalssuck.blogspot.com
Winner:
CASSOWARY



His Fossil Mammals:
http://mammalssuck.blogspot.com
Winner:
GIANT GIRRAFID
illustration from the book Kameno doba by Jovan Zujovic (1856-1936)
 So his Final Four and Winner are:
http://mammalssuck.blogspot.com
He had lots of good quotes when picking these but my favorite was his rendition of the Cassowary. I showed him a picture and it became the "Giant Squash Bird!" and would go WAM WAM WAM on all the little guys. And so I present this image to you:

GIANT SQUASH BIRD! Cassowary destroys the competition to make it to the final match-up!
All images mine, except for Cassowary which is from http://a-z-animals.com/animals/cassowary/pictures/4128/


"Echinda because Babirusa will stop on spines and go OW OW OW ::hopping around on one foot like a cartoon character" 



My prediction: In a Cinderella story for the ages, Perry the Godzilla Platypus will claw his way into the final four

 and then be mercillessly mauled by the Polar Bear who goes on to win it all!

My bracket:


We are both 7 out of 8 right now, how are your brackets looking? Leave a comment and let us know!

Monday, March 10, 2014

Soybean why you torment me so..

Not only are you slow growing, but you refuse to stop intertwining with your neighbor. Every day I come up and gently remind you and remove you from your neighbor. Yet you never listen. How am I supposed to be sure you're selfing when I keep catching you snuggling with the neighbors! I need you to self so that I can get nice homozygote lines for my planned experiments. Any co-horting you do now could jeopardize my plans. I know quarters are tight and nights are cold, but please for my sanity, please stop the intertwining!
Evil twisting soybean

Friday, March 7, 2014

Mammal March Madness!


I have just learned of this awesome animal based March Madness bracket event occurring called Mammal March Madness 2014 and it looks amazing. They have set up 4 divisions of, mostly, mammals to simulate battles between in a NCAA bracket-esque event. The four divisions are:
  1. Social Mammals (they get to fight in their groups!), 
  2. The Who in the What Now? (mammals most of us do not know, learn something new!), 
  3. Fossil Mammals (back from the past!),
  4. Marine Mammals (my favorite group!).
Each division features one non-mammal: army ants, cassowary, megalania and oceanic white tip, respectively. In addition to this being one of the most awesome brackets I have ever seen, it is turning out to be a fantastic learning experience. Boo and I are both doing our own brackets and we have NOT picked the same, cannot wait to see who is right.

Tonight, we sat down and made our picks for Social Mammals and Marine Mammals. We got out his Smithsonian The Animal Book and my laptop for anything not covered in his book. First things first, I taught him how to do research. Look at the name of the animal on the bracket (we are using the common name bracket, not the Latin names.. this year) and then look it up in the Index at the back of his book. If it was there, we would flip to the page and read about it. If it was not there, I would get out Google and find it. Then we would talk about what factors might be important to the individual battle, such as animal size, speed, strength, social group size, etc. Then he would write down his prediction and move to the next set.
Research, research, research!
By this process he learned how to research, how to compare, and now he has predictions that during the actual 2014MMM will be tested. After each round we can talk about why each animal won and, for the ones he got wrong, what factor he might not have considered. This is kid friendly science at its finest! We have already learned about a few new animals and new facts about animals we already knew and loved. I HIGHLY encourage everyone to try this!

His Social Mammal prediction:
 Copyright: http://mammalssuck.blogspot.com
Bracket courtesy of: http://mammalssuck.blogspot.com

Social Mammal Winner:

MANDRILL

His Marine Mammal Prediction:
Bracket courtesy of http://mammalssuck.blogspot.com

Marine Mammal Winner:

OCEANIC WHITE TIP SHARK


 


Some of the better quotes that came out of our research tonight (pictures provided in case you are not familiar with that animal):

"HAHAHAHAH Musk ox will just pound that little tail-less squirrel"

"Wolf, cause it can run faster and the ox is low and slow so the wolf will run it down and jump on it and eat it"

"Mandrill will beat the wolf because it can bite and break wolves bones."

"Narwhal will go ::tosses his head into my stomach:: with its tusk! Easy."

"Walrus tusks into heart Orca dead."

We will do the other two brackets over the weekend. This is going to be fun!! Play along with us! Post your picks or pictures of your picks in the comment section!

Wednesday, March 5, 2014

Experiments We Love

Last weeks tips for young scientists seemed to be a popular post, so I thought I would write out directions for a few of the experiments we enjoy. I picked things that need none to minimal special equipment, most of which can be found at your local grocery store.

 Color Changing Volcano 



This was his favorite experiment from 3 til very recently. It is really great for little kids who are
still exploring what color mixing will do, not to mention it makes a big mess so how can you go wrong :)
Steps:
  1. Put water and baking soda in one container, add baking soda until no more will mix in with the water. 
  2. Put white vinegar into another container
  3. Add different primary colors to each container. I've done it before with lots of test tubes so that he had lots to test. 
  4. Depending on age/ability of your child, give them the test tubes and let them pour into an empty container or simply have them observe as you pour things together.
  5. To mix it up: They can measure out different amounts of the vinegar/baking soda. What ratio gets the best explosion? 50/50, more vinegar, more baking soda? What happens when you mix all 3 primary colors? Can you get different shades of purple/orange/green by adding different amounts of red/yellow/blue? 
How it works: Vinegar is an acid and baking soda is a base, when you put acids and bases together you get what is called a neutralization reaction: Acid + Base --> Water + Salt. In this case, carbon dioxide is a byproduct of the neutralization reaction which makes the bubbling.

Pineapple Enzyme Exploration 

Pineapple, like all living things, is full of enzymes. Enzymes catalyze the reactions inside a cell, they make things go faster and cost less energy! Because enzymes do things, you can actually see the results of their work.
Steps:
  1. You need fresh pineapple, canned pineapple and 3 premade Jell-O cups. 
  2. Cut the fresh pineapple into small cubes. 
  3. Open the 3 Jell-O cups, leave one with out any pineapple (control). Add the freshly cut pineapple to another cup and to the last cup add the canned pineapple. Observe the Jell-O.
  4. This takes a while, after about an hour you can see a difference but after several hours it becomes very apparent. A good time to do this is before school, then when they get home the kids can rush to see the results.
  5. Another variation is to make Jell-O and put 1/3 in the fridge without pineapple, 1/3 with fresh pineapple and 1/3 with canned pineapple and see which ones solidify. 
  6. To make this into more of an experimental setting versus a demonstration setting: ditch the canned pineapple and let the kids treat the fresh pineapple with various things to see what will de-activate the enzymes. Some examples: Microwave at various lengths, freeze it, treat it with chemicals, like bleach (base) or vinegar (acid) that change the pH. 
How it works:  Pineapple has bromelain which are protease enzymes. Protease are enzymes that break down protein. Jell-O is collagen, a protein. When you combine bromelain with collagen, the result is broken down proteins. Lots of different factors can impact enzyme activity, but temperature and pH are two big ones. When they can pineapple they Pasteurize it. AKA heat it until all the potential disease causing critters are dead. The fact that fresh pineapple can "melt" the Jell-O and canned pineapple cannot is because not only does heat kill bacteria, it can also denature (or break) enzymes.

Goop or Ball

This does require Borax, which is a laundry booster/cleaner and can usually be found in the stores though it may take some searching. Borax is the only part of this that you probably do not already have laying around the house.
Steps:
  1. Gather the materials: Glue (we use cheap white school glue), Saturated Borax (put warm water into a bottle and add Borax until it refuses to dissolve, that's saturated), Water, Plastic bowl, Stir stick, graduated cylinder/measuring cups, and if you want your goop to be colored, food coloring.
  2. Measure out equal parts glue and water and stir together (we do 50ml). 
  3. Add food coloring and stir, if you want colored goop. 
  4. Add Borax solution (30% of combined glue/water mixture, we use 30ml). Stir and you should immediately notice the chemical change occurring.
  5. Play with your goop! It should be sticky and stretchy
  6. To make a bouncy ball, you need to add less water to the glue mixture so that it becomes thicker. This is messy, but fun, you have to pick up the mixture and work it in your hands to become the ball shape. 
  7. Have the kids experiment with different proportions of water/glue/borax to see how the changes impact the reaction. 
How it works: Borax when dissolved releases a borate ion. This ion interacts with the polymer (polyvinyl acetate) in the glue, making a polymer-borate-polymer bridge and forming an even more complex polymer with greater structure than the glue.

What Keeps me the Warmest? 

The idea of insulation is one that us as adults are familiar with, we either praise or curse the insulation in our houses, we like can koozy's when tailgating, travel cups for coffee, etc. But not everyone thinks about the fact that coats/sweaters/etc. are insulating our body from the winter weather. They serve the purpose of keeping heat inside our bodies, not letting it escape into the environment. And there is a fairly simple way to let kids investigate this, though it REQUIRES adult help due to the use of boiling water! You will need a thermometer (we used a digital meat thermometer), jars/glasses that can handle boiling water, boiling water, a timer, and several articles of clothing to test.
Steps:
  1. Boil water (ADULTS) and measure the temperature. 
  2. Pour water into the jars and close lids if there are any (ADULTS). 
  3.  Put clothing articles over the jars. For his K science project, we did his coat, his hat, a sweatshirt, and nothing (control). 
  4. Wait a specified amount of time. 
  5. Measure the new temperature in each jar and compare it to the first (adults should monitor this as the water inside the jars will still be hot). Which stayed the warmest and thus had the best insulation? 
  6. This is a really versatile experiment because you can test a lot of things, from packing material to clothing. You could run a time course, take the temperature every X minutes for Y time.
How it works: The second law of thermodynamics  tells us that energy only flows from hot to cold objects and the heat will continue to flow until equilibrium (equal temperature) is reached. The articles you place between the hot water and the cold air act as a barrier to the heat transfer and slow the process down. The better insulator the object is, the longer it will retain its heat.

Monday, March 3, 2014

Did You Know?

Qualifying exams continue to creep closer and my studying continues to progress. I am actually making progress to catch back up to my original plan of study, just over a week behind. I should be able to close that gap before bed tonight. But I thought I would take a break and provide some highlights from all of the studying I have done this past few months.

  •  Photosystem II is still the most awesome part of biochemistry and completely unappreciated in your basic biochemistry textbook. Photosystem II captures light energy, and passes the excited electron down the pathway like a hot potato! Did you know scientist's can actually measure how quickly a single electron is passed from QA to QB?
  • Protein degradation is a big deal to cells, it keeps unwanted/damaged proteins from building up and allows their building blocks to be recycled. When you consider that cells need to change rapidly to changing conditions, the need to breakdown no longer relevant proteins makes sense. Did you know that some proteins have a half-life of only 30 seconds? 
  • Complex III of the electron transport chain is just photosystem II in disguise, just not as cool since it does not capture light. But did you know that what it does do is help balance the electron flow by breaking up a pair of traveling electrons into two individual and independent electrons?
  • There is a set code that dictates how genes stored in the cell are turned into proteins that do the work. This code is based on 3 letter words called codons, composed from the 4 character nucleotide alphabet (A, C, T, U). But did you know there is actually a fifth character (I), that allows a "wobble" pairing which makes the code more like guidelines than actual rules. 
  • The human genome is composed of 23 chromosomes with a total genome content of about 3.3x10^9 basepair long. But did you know that about 1% of that is used for encoding protein?
  • While not really a cool fact, did you know there is a lot of math in biochemistry? So many pages devoted to free energy, reaction kinetics, Hill equation, equilibrium... been a long time since I worried about so many numbers!
There you have my brief thoughts and fun facts picked up from studying. If there are any of these topics you would like explained in more detail in a future blog post, leave a comment or drop me an email!