Wednesday, April 30, 2014

DFPM: Pizza

Dear Future Professor Me,

You remember pizza.. pizza is AWESOME! It's gooey and delicious, customizable and often delivered right to the door.  Seriously, look at that.. how do you not drool!


You know what is even more amazing to students? Free pizza! Something to keep in mind for building camaraderie in the lab.

Monday, April 28, 2014

Tricksey Transposons

Three weeks until quals.. It is sneaking up so quickly! I am feeling completely unprepared and panicked. I am almost done with my first read (making notes from every page of the assigned reading list). The last topic is transposition! So I figured I'd combine studying with blogging tonight and bring you the featured topic of the day.

Transposons are mobile DNA elements found within genomes of all organisms ranging from the simple, like bacteria, to complex, humans for example. They can literally jump to a new spot in the genome! Transposons make up half of the human genome and are considered to be the major force driving mutations. Transposons are divided into families based on their movement mechanics and we'll take a quick look at the three types: DNA transposons, virus-like retrotransposons ad poly-A retrotransposons. 

DNA transposons are composed of target-sites and terminal inverted repeats flanking the transposable element which often contains genes that participate it's movement. In the following diagram, XX will represent the target sites, > < will represent the terminal inverted repeats and e will be the element.

XX>eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee<XX

Within the element is often a gene to encode transposase, the enzyme responsible for moving the transposon around. DNA transposons are moved via cut and paste. The transposase binds to the terminal inverted repeats, bends the DNA so that these sequences line up and snips it out. Then it snips the target DNA and inserts the transposon element without expending any cellular energy. Now the genome has changed in 2 places. The original location of the transposon now has a deletion in it and the target now has an inserted transposon. You can imagine how this might impact the organism.. it could remove an important section of the gene, it could drop an element into a gene and make it nonfunctional or it could have no impact whatsoever.

Virus-like retrotransposons are organized with two long terminal repeat regions, that contain the target-site and terminal inverted repeats similar to DNA transposons, surrounding 2 genes: one for integrase and one for reverse transcriptase. To diagram this we will use L for the long terminal repeats (with X and > < inserted), I for integrase and R for reverse transcriptase:

LLXX>LLLLLLLLiiiiiiiiiiiiiiiiiiiiiiiiiirrrrrrrrrrrrrrrrrrrrrrLLLLLLLLLL<XXLL

Reverse transcriptase is an important and unique enzyme that runs backwards from the central dogma of genetics. Reverse transcriptase can change an RNA strand into a DNA, they go backwards and thus are "retro." When a retrotransposon is trascribed, the RNA is recognized by reverse transcriptase and turned into a special type of DNA called cDNA. Integrase targets cDNA and integrates it into the new target site of the genome. Since the original retrotransposon is not removed, simply transcribed into RNA and then changed into DNA and integrated into a new location in the genome this can be thought of a copy and paste method.

Poly-A retrotransposons, are the last major family of transposons. Unlike the previous two families, Poly-A retrotransposns do not have terminal inverted repeats. Instead the target-sites flank a 5' UTR at one end and a 3' UTR which has a lot of A's (adenine, one of the 4 nucleic acids that make up DNA) after it on the other. Inside the UTR are 2 protein encoding ORF's. To diagram these, XX still is the target site sequence, 5 will be the 5' UTR, 3 the 3' UTR, and 1, 2 for the OFRs.

XX5555555555111111111111112222222222222333333333AAAAAAAAAAAAXX

The presence of the 5' and 3' UTR, plus the repeating A sequence, makes poly-A retrotransposons appear gene-like. They move in a similar fashion to genes. The DNA is transcribed into RNA which is then translated and produces its two proteins. These proteins then bind to the RNA and move back into the nucleus. The protein-RNA combo binds to the target DNA, snips it and uses the poly-A sequence to attach to the DNA. A reverse transcriptase function of the protein then creates new DNA based on the RNA which is attached to the genome. A few fancy steps later, the RNA is degraded and the brand new DNA has been inserted.

So take home message: Genomes can be rearranged spontaneously via transposons. There are 3 types. DNA transposons jump via  cut and paste method. Retrotransposons move via an RNA intermediate in a copy and past method. Poly-A retrotransposons more closely resemble genes with their asymmetric ends and move via an RNA intermediate in a copy and paste method.

Friday, April 25, 2014

Mommy First Aid

Whenever I have taken a first aid course, the first thing they always emphasize is SAFETY. It does not help anyone if you go into a dangerous situation and now there are two people in need of assistance. If someone has succumb to a fallen power line and there is water between you and them, call the professionals so they don't have to get you both. If someone is drowning and you are a moderate swimmer, get HELP don't swim out there cause they will drag you down with them.

I never realized as a kid that this is what my parents were doing when they sent me to my room to think about what I had done. They were taking a "let me settle down so I do not go totally ape shit on you" approach. In a way, it was parent first aid.. take time to be sure the rescuer is not going to make the situation worse before tackling it.

Now years later as a Mom, I have battled the ever present Mommy guilt of not being practically perfect in every way. I tried to interact with the problem head on and more often than not I learned I was ill-equipped. He would scream, I would try to be patient and he would scream more and I would start to scream and then we are both screaming at each other and NO one is safe! But I would feel incredibly guilty, or like he was "winning", if I walked away and ignored him. So I decided to be insane.. you know, doing the same thing over and over again and expecting different results!

Since we've moved for grad school, we've had some doozies of conflicts. And I know his lashing out is because we've moved and he is not getting the amount of attention he used to get before the move when I only worked part time and we lived with my parents. After a few blowups that ended with me crying cause we had both screamed things we didn't mean, finally, I started walking away or sending him to his room. Most of the time because we both need it, but sometimes because I need it.

I've also started telling him instead of stuffing it down and trying to be Perfect Mommy when I'm getting overwhelmed and then it building up until I snap. I can relate it to when he gets overwhelmed or bothered by something and how that feels to him, Mommy is feeling like that and just needs a little quiet. Most of the time he respects it and lets me have some time. It is slow progress and we both lose it occasionally but at least if I can start recognizing when I am overwhelmed and communicating my feelings with him, perhaps he will imitate this and be able to communicate when he is overwhelmed.

I know I am not the only one who feels overwhelmed with her kid. But most Mommys (myself included), will push forward and put kids needs above their own on a regular basis. Even if it might cause more drama later. We often ignore our own needs and/or feel our needs are insignificant compared to our kids. And while that is often true (kids need a LOT), I think there are also times where taking a break is required. There needs to be more mommy first aid going on.. so theses are my suggestions for a Mommy First Aid Course:

Mommy First Aid Flow Chart

1) Are they in any immediate danger? If no continue to step 2, If yes, interact immediately
2) Are you calm/ready to engage? If no continue to step 3, If yes, interact immediately.
3) Take a few minutes to clear your mind. Relax/Let it go/walk away/get a cup of tea, whatever you need for YOUR sanity. Trust me a few extra minutes is not going to irreversibly hurt them.
4) Ok calm and ready to engage now? If no, repeat step 3, If yes, interact immediately 

Wednesday, April 23, 2014

Sometimes you get the bear...

sometimes the bear gets you! First off, I must apologize for missing Monday's post. Boo had some routine tests at the hospital that I thought would be no big deal that turned out to be absolutely exhausting/stressful for both of us. So I did not get anything written.

I learned I have the most awesome PI adviser to ever be a PI adviser! After Boo's tests, he ended up not going to school yesterday. I needed to start a bacterial culture or I was going to be insanely behind this week. So I went in and got my stuff prepped then told Boo he had to SIT right there and not move until I got back (didn't want to take him back with all the cultures!). My PI goes "Hey I'll sit with him and he can tell me all about his cool toy there!" and merrily drops into the chair next to my kiddo for the next 5 minutes while I ran and inoculated my cultures and put them on the shaker. While it was only for a few minutes, I really really appreciated it! It's the little things that can help out in the largest ways.

After lab, I took Boo to the zoo for us to de-stress. We ran into one of my students at the Zoo, working in the aviary. She had a bird on her shoulder and put it on Boo's shoulder. He was so excited as the bird (Casey) crawled around his shoulder and up onto the top of his head where it perched for a while until he was like ok, take it off. Then he got to feed some of the little birds. It was just what we needed. I got the bear!

This morning I came in and started my plasmid extraction. Going merrily along through the first six steps when I pulled them out of the centrifuge, there was hardly any pellet. Usually at that step the pellet is huge! Consulted with PI and realized I forgot to add the lysozyme.. so my bacterial cells did not fully break apart meaning most of my DNA was in the bleach water now.
Annnnd had to start the cultures over, so the quick run into lab with Boo did not help me stay on pace. My own idiocy ended up setting me back a day anyway! Yep, bear got me.

Thursday, April 17, 2014

Anxiety

Boo is a highly anxious child. He has therapy at school to help with his high anxiety and most of it is separation anxiety. Unlike some, I've never lived with his Dad. We've always had a long distance relationship and for the first four years of his life, Boo never spent the night at his Dad's without me. Then things went sideways and the current custody schedule with week/weeks at a time at Dad's without Mom hit.. then he started school.. then we moved. Each year since he turned 4, something has happened that decreased the amount of time he spends with me. While I, as an adult, understand that this is the natural progression of growing up, Boo, as a highly anxious kid to begin with, feels like the separation is to long and what if Mommy doesn't come!

I have always tried to reassure him that Mommy always comes to get him. Yes, he goes away, to school, to after school care, to his Dad's, etc. But Mommy always comes and gets him. She has and will never abandon him and will always be there. And until this last trip that was 100% true.. then spring break hit. I was supposed to pick him up at the end of the first week of spring break. On my way to get him, I had that awful, back exploding, unbearable, unmistakable pain of a kidney stone attack during my drive. I tried.. oh how I tried.. but eventually I started shaking and vomiting and could drive no longer. I pulled over, called my parents, called my ex and checked into a hospital. Now I have a FANTASTIC family.. seriously.. they all got up early on their Sunday morning and rallied quickly. My brother and his wife went to get Boo, my dad and his dad came to get me, my mom and my grandma stayed to hold down the fort in case Boo beat me back. As emergency situations go, things went very smoothly!

When Boo first got back, he had a lot of fun with my brother/sister-in-law. They took the scenic route and bought him super sweet binoculars. He said he was great and asked how I was and snuggled for a bit then went to bed. Everything seemed fine. Then a few days later, he was crying in bed and I asked him what was wrong. He said he was so scared something was going to happen to me. He does this sometimes, and I reassured him that I was going to be fine, nothing would happen.

Yet, for the first time ever, he was able to look at me and rebuttal it could since it just did. I couldn't come get him because I was in the hospital and he was so scared and it was not what he was expecting and plans changed. Must admit, took me a second to catch up. He caught me.. I'm not invincible! Drat. This led to his fear that he will be alone if something happens to me since we don't live with grandma/grandpa anymore. It was a late night, but we talked and it seemed to help. I wish I knew how to help him with his anxiety.

What about my readers, any tips you've found that help relieve anxiety?

Wednesday, April 16, 2014

DFPM: When Things Go Wrong

* Note This is the first in what I hope will be a series of letters to myself as a future faculty member, hence DFPM - Dear Future Professor/PI Me. As I run into things as a graduate student, or see things around me, that I want to remember something about it when I get to be Professor/PI. *

Dear Future Professor Me,

When your new graduate student suddenly realizes she completely mismanaged her time, is trying to cram a weeks worth of work into two days and fails miserably, remember.. you've been there. Sometimes the noobs do not think all the way through things. Crack a small smile, nod and say "ok that happens." It's amazing how much that simple acceptance helped calm nerves.

She is probably feeling incredible stupid. A feeling you might not remember now being a hotshot professor, but you have been there. Remember that time you had to toss out a whole flat of Arabidopsis because you did not get the plasmids made in time? Good, hold on to that feeling. Pass on some reassurance.

She is probably feeling really nervous having to tell you that she messed up. Her timing was off, things took longer than she thought, her experimental yield was low, whatever the reason was she might not think it is a good enough reason and be second guessing her ability to plan.

She will feel very relieved, even if she notices the hint of amusement, when you nod and understand. Don't let it become a habit but the first time your new grad student completely mismanages time in the lab, shrug it off. You'll be amazed how grateful she will be and it adds to your cred!

Sincerely,
Lowly Grad Student Me

Monday, April 14, 2014

Wait.. Finals week is WHEN?!

The semester is FLYING by right now.. Only 3 more weeks of classes and then finals.. more importantly (and terrifyingly) quals are exactly 5 weeks away. Soon I will have completed my first academic year as a PhD student. I have 2 more lectures + exam to write/give in the course I am teaching, 1 more talk to give in my seminar course, and 6 more chapters to finish a first read of the reading list for quals, THEN I have to study all of my notes from the 5 textbooks. Oh.. there it is.. the panic setting in.

AHHHHHHHHHH!!!!!!!!!!!!!!!!!!! ::makes a loop screaming around the apartment::

Ok better.

Things in the lab have gone all ka-blewy last week and fall out continues this week. And with quals 5 weeks away I cannot plant anything so my experiments will have to wait until June. Now I return to my reading. Sorry the post is so small today

Friday, April 11, 2014

Lego Egg Hunt!

such as this classic dress + hat!
Easter is rapidly approaching. And I mean rapidly, at least to me it feels like it is arriving at warp 10! With qualifying exams on the way, plus my course presentation this morning, lab work I am just now starting to think about Easter!

As a kid I loved everything about Easter. Pretty dress, pretty flowers, a basket full of candy and an egg hunt of awesome. Boo, being a boy, has lost out on the dresses and after our first few Easter sugar highs I decided I disliked that much candy. Candy in the basket, candy in the eggs.. it's enough sugar for the entire year all crammed into a day!So a few years ago I decided to start something new and boy was it a hit. 

Lego Egg Hunt!

I buy one nice set, open it the night before and stuff all the Lego pieces into the plastic eggs! I have 3 larger eggs to accommodate some of the larger Lego pieces and the book + huge pieces go in his basket with a small selection of sweets. The big tip here is: know how many eggs you stuffed!! If all the eggs are not found you do not have all pieces for the set! Requires a little bit of organization to be sure I know where all the eggs are and how many total before he starts working on the set or else we will have a panic/frustration meltdown because a piece is missing. 

If you're looking for an alternative to candy in your eggs, this has been a huge hit for the last 2 years and he will not stop talking about how excited he is to see what set will be in the eggs this year.

What are your Easter traditions? Do you have any other non-sugary ideas for Easter egg hunts? 

Wednesday, April 9, 2014

Seafloor Explorer


Last week I got an email from Seafloor Explorer saying that they were 81% complete with just over 2 MILLION images analyzed via crowd sourcing after 18 months! And they said they have another batch ready to upload once this first set is finished. That's incredible!

I LOVE Seafloor Explorer.. it is my go to relaxation website. By now you might have guessed based on the name that is a website that lets you examine images from the seafloor. For the last several years, they have been using a camera pulled from a ship and taking about 6 images per second across the Northeast Atlantic. Since they can get thousands and thousands per run and they have been doing multiple runs a year, they end up drowning in image data. So they decided to try crowd sourcing, putting the images online for citizen scientists to examine. There is a tutorial and you learn to identify the ground cover (sand, shell, gravel, cobble, boulder) and how to label certain organisms (scallop, fish, crustacean, seastar).  Scallops and seastar are by far the most common organisms I see when I analyze. And a lot of the images have few to no organisms. 

Or sometimes they have a lot of organisms but they are all in one category like the two below.


But there are those pictures you come across that make you gasp and marvel like this beautiful ray!

Or this

So if you have some time and want to help map habitat type and species distribution across the Atlantic, check out Seafloor Explorer! I find it very relaxing and fun. Boo also enjoys curling up and helping me spot organisms, he gets bored if we run into a stretch of empty images but on the flip side gets incredibly excited when we find anything.

What do you think about crowd sourcing science like this? Have you ever tried these type of citizen projects? If not go check this one out and let me know how you like it!

Note: all images are copyright Seafloor Explorer

Monday, April 7, 2014

Troubleshooting Terrors

I've been learning to transform protoplast and my first experience went smashingly! However, the next attempt had a very low survival rate (~50%) and I could not find a transformed cell! All of my solutions were freshly prepared, so it's not an age/storage issue. I used the same plasmid DNA so they should have transformed. Our microscope is behaving strangely, the computer died so I cannot use the software and have to do it by hand. This should not impact their survival rate but could be giving me false information on the transformation rate. I started another one tonight and I'm going to do a GUS assay and Western blots on the samples I collected last week + tomorrow to see if it's just the microscopy that is the issue or the actual transformation.

Even in established protocols, things go wrong. Sometimes you know what and how to fix it, sometimes its completely random. For example, a few weeks ago, I did a PCR with different primers but the same DNA as my previous one.. and got no reaction. But I realized that I had used the same protocol and not adjusted for the fact that I had different primers. With PCR, temperature is critical and every set of primers has it's own "magic" temperature. So I had screwed it up! Operator error, my favorite kind! Straightforward troubleshooting and we move on.

But then I did another PCR using the exact same DNA that I had amplified before, primers that I had used before, and our stock master mix. Did not work, no bands no smears, nothing. I scratched my head because everything had been used in previous reactions so I could not understand what had happened. I checked my protocol, the temperature was correct, the cycles were correct, everything checked out.
I tried it again and replaced the ONE thing that had not been proven in other reactions, the water. Now we use special purified/filtered water that has been autoclaved so that it should have NO contaminants in it. But it also a shared commodity so sometimes they go bad before they are gone. Boom perfection. Seriously.. water.. WATER?! This is how sensitive PCR can be.

Thankfully, I have been doing PCR's routinely since year 1 Masters, so I know how to troubleshoot and often guess correctly on what might be causing my problem, even if it is something as silly as water. These protoplasts are new to me, I'm going off of a thesis of a previous lab member and 2 published papers and while there are some helpful hints, it is far from step by step. And no current lab member has done this before, so I have no one to go ask. Even my PI is only vaguely familiar. So I get to play troubleshooting in a situation where I know very little. Which means lots of reading, lots of guessing and simply trying stuff until it works!

Troubleshooting, especially in the situation where you have NO ideas, is a great skill to learn. It is also a thing I detest! I am a control freak, I can admit it. I like to have a clue, a plan, a road map! When things do not work, that should, when my road map looks more like Daffy Duck's from Duck Dodgers and the 24th 1/2 century's map, I freak out a little bit. Ok, a lot. I actually had to just go home last week after my transformation went so awry. I could have tried to start another one immediately, but I wanted to spend the weekend trying to find a new plan. I used the time to study for quals (which are rapidly approaching *PANIC*). Over the weekend, I did read and I think I came up with a plan, but only time will tell. For now I guess I will just have to try and channel Mr. Tim Gunn and

What do you do when your road map suddenly goes from a beautiful path to insanely complicated? Does it drive you insane or are you able to adapt on the fly?

Friday, April 4, 2014

Boxes make the best toys

So apparently I've been doing this whole parenting thing wrong.. instead of spending money on toys I should treat Boo more like a cat and provide him with boxes. Lots of boxes...and tape... lots and lots of tape.



My parents saved a lot of the boxes I used for moving last summer. Boo has taken to creating vehicles out of the cardboard boxes. The ideas are 100% his, he will get help with cutting or architecture (some of his ideas are WAY to complex for cardboard!) but this is all child led. And it has been insanely awesome, and frustrating all at the same time.. but more on that later :) 

His first project involved a single box, he cut the sides off of it and used them to make rear view mirrors, a siren, pedals and an iPod holder inside the car. It got decorated with crayons and is the Jaguar 360 he took to the drive-in ;)
Boo doing cat imitations
His first creation was adorable. But he couldn't stop there. The next creation was a semi truck. Seriously.. cab and a trailer that can hook/unhook. He cut a bunch of flaps off boxes and taped them together for the trailer and taped two boxes on top of each other for the cab. It has a door so he can get in and out, a bowl for a steering wheel, a working windshield wiper, and screws to attach the trailer. During the decoration process it acquired a siren and became a police truck.
Finished, fully colored, semi police cab, there's
a computer inside for tracking bad guys
screws to attach trailer




Next, he decided he needed a race car. One where you could slide your feet in just like the Formula 1 cars.  This one he just HAD to use paint.. my Mom is such a good sport and let him paint on the table!
He also used a tea box and a paper towel roll to make a shifter inside each of the vehicles..
He worked on this the week of spring break while he stayed up at my parents for spring break. I got to help the first weekend and then see the completed fleet when I retrieved him.

As you can see this project has been full of awesome. It was also frustration laden when he had ideas that were beyond what cardboard could accomplish. He also got fixated and would tantrum if he could not work on them but have to do something else, like eat or sleep. There were several moments through this process that I wished I had never let him have the first box. But he really enjoyed it and each one got more and more detailed, especially the drawing he would do to complete the interiors. It was a great creative outlet. I wish I could help him manage the fits a bit better, but I think having to work through them over little things will help in the long run for the bigger things.

What about you guys, any creative usage of boxes in your house?  What's the best creative masterpiece using common items you or your kids have ever come up with?

Wednesday, April 2, 2014

FAQ: How do you manage!?

I get asked a variation of how do you manage to do all of this a lot when I say I'm a PhD student single Mom. My answer is usually.. depends on the day! When I stop and think about it though, I am able to pull out a few common threads that help me manage all of this insanity.

Motivation 


I want this PhD and I want it badly. When I was 8 I decided I wanted to be a marine biologist, hence my MSc in marine biology and biochemistry. As I grew up I discovered how much I love to teach and decided it would be a PhD and Professor J for the rest of my life. Then I got tossed off track by the curve ball called Boo. And it took a while but I'm back and I really believe I CAN do this and I really want it. Even with the horrible sadness that I battled when Boo was gone, I showed up ready to work and got my stuff done efficiently. To balance everything requires an important life skill...

Time Management 


Lists are my best friend. I have so many different things going on at any given time that I have to be organized and manage my time properly. This semester for example: I'm teaching, growing transgenic soybeans, trying to create a simple genotyping method for selecting my soybeans, making Arabidopsis protoplasts, selecting Arabidopsis transformed seedlings, studying for qualifying exams, reading literature to figure out where to go with my project, taking a seminar course, helping Boo with homework, playing with Boo, blogging, and general housework/adult crap. I try to make a list every week that covers the weekly MUST DO and the weekly like to do for school and one for home. As the visual aid shows above, there is not enough time to get everything done so sometimes you have to.....

Let Stuff Go


There is NOT enough time to get it all done.. it's simply impossible. This is where priorities really come into play. There is, unfortunately, something to be said for due now, do now. A lot of weeks my apartment does not get vacuumed, laundry will not get done until one of us is down to our last pair of undies, if no one has vomited the bathrooms may or may not have been cleaned for a few weeks and even a shower is a luxury some nights. I consider it a win if I get dishes done each night. The week of my lab meeting I get very little studying for quals done because I need to get my presentation in order and the house is a guaranteed disaster zone. Some nights I get nothing done because Boo is sick or just needs extra time and he is my first priority. Second priority is lab with teaching coming up right behind it. Having a perfect apartment, doing perfect research, being a perfect Mom, being perfectly groomed, will never happen. Perfection.. let it go!

I strive to do the best I possibly can, work as hard as I possibly can, but also cut myself slack to know I'm going to screw it up. I'm going to not hit a deadline, I'm going to neglect Boo for a few days when school is nuts, I'm going to go longer than I should between cleaning my house and that is going to be OK! Sacrifices have to be made. After all...
 How about you? Any good ideas for balancing and coping that work well in your lives? I'd love to hear about them in the comments!