Repetition

                This post will be my final one for 2011, which became my inaugural year as a blogger.  I’ve enjoyed being able to share different things about science with all of you, so thank you for reading!  I also like having a place to explain what working in a lab is like every single day.  It’s not quite as exciting as Abby from NCIS would lead you to believe (like - at all), but it’s certainly not bad!  We have the freedom to make our own schedules, we sometimes gather up every item in the lab that matches a post-doc’s shirt color and take pictures, we put dry ice in closed containers to create bombs, we can make water balloon gloves, and we are regularly subjected to meetings about which we know very little but need to act excited.  Most of us are poor actors on that last one.

We do, however, have moments of sheer excitement.  These moments typically follow the endless repetition that is troubleshooting one experiment.  

Let’s review the speed at which scientific research moves by reviewing the breakdown of 2011.

January 2011 – May 2011: completion of one experiment

June 2011 – August 2011: exploring different areas of where my project could go; very little headway on anything.  Cue me having a complete meltdown.

September 2011 – November 2011: Completion of a second experiment

December 2011: Mild troubleshooting, writing a paper

                Okay, look that over.  Two experiments = one mini paper’s worth of work.  It took me five months to complete the first experiment and four months to complete the other.  Yes, they were large and multi-layered experiments that required precise work to get them running properly, but that’s a total of nine months.  I could have grown a functioning human in that amount of time. 

Science is patience.  

And alcohol, which serves to dull the insanity that inevitably follows endlessly repeating the same experiment over and over again.

Okay, let’s discuss why it takes so long.  I’m going to focus on my second experiment and explain, in loose details, about what took three-four months.

Let’s say this experiment required four things: A + B + procedure = results.  This means that I mix A and B together, followed a particular procedure and then was able to see my results.  

Oh man – are you ready?  I first had to make A and B.  Making them is no small feat and required a few weeks worth of work to make them and prove that they were made correctly.  Okay, that’s three weeks there.

Next, I had to perfect the procedure for mixing A and B together.  This means that I take a stab at how the experiment should work.  Then, I look at my results.  Inevitably, they are not great the first time around (if they are, well, you have a horseshoe stuck).  So, I need to tweak it.  For example, I say “Well, maybe I let A and B sit on ice for too long.  Let’s try the procedure again but let A and B sit for a shorter period of time.”  This means I go back and do the same procedure again, but maybe I’ll let them sit for 10 minutes or 15 minutes.  I’ll probably do both and check out my results again.  And I’ll tweak more.

Seriously, perfecting the procedure of an experiment can take anywhere from a few days to a few weeks or even a few months!  For me, perfecting my procedure took nearly two months.  Each and every part of the procedure needed to be tweaked and optimized.  This included how much of A and B were added, the length of time things were mixed together, the time I spent working up the experiment, how I viewed the results, the liquid I used to mix A and B, etc.  Ohmygod.  I’m tired just thinking about it.

The best part?  Sometimes you spend so much time perfecting your procedure that you run out of A or B.  This means you must go back to the beginning and make more.  Sure, let’s add another three weeks!  What’s the difference?

When you finally have perfected your procedure, have enough A and B, and are getting consistent results with your procedure, then you do the real experiment.  Up until now, you’ve been working with controls.  Controls are experiments that follow your exact procedure but you know precisely what the outcome will be.  The real experiment is when you follow your exact procedure and add in ONE extra thing.  If your results change from your controls, then you know it was due to the ONE extra thing.  From this, scientists can draw conclusions about that ONE thing.

For example, let’s say A and B will always bind each other.  We have perfected our procedure to show over and over that A and B will bind each other if and only if both are present.  When we do our real experiment, we add in a small molecule with A and B.  When we see our results, we notice that A and B no longer bind each other when the small molecule is present.  This means that the small molecule inhibits their interaction.

For me, running my real experiments took about two weeks.  

This all adds up to three-four months worth of work.

Sigh.  For these reasons, scientists are constantly working on different projects simultaneously.  If one experiment becomes stuck, the scientist still has another project that may be working and yielding publishable results while the other project is stalled (and vice versa).  Keep this in mind when you hear how much money has gone into research over the course of time and ask yourself why science doesn’t move faster.  All of our results need to be reproducible and above reasonable doubt for working correctly.  The time spent in developing and implementing a solid experiment is necessary.  Also, think about the slow madness that envelops a scientist when they march into work for the third month in a row to perform the exact same experiment with one small difference.  There are many reasons why scientists are sometimes called “mad.” 

A few weeks ago, my boss brought in his son for a Christmas party.  The four year old was walking around the lab looking at our things.  At one point he asked, “Dad, why does Mark have so many of the exact same bottles?”  His father said “Because that’s what science is.  Doing the same thing over and over …and over again.”

Here's to a happy and healthy 2012!

REFERENCES

Me, myself, and I

Funnies.

                It’s the end of the year.  Let’s just have some fun.

                During graduate school, I found Ph.D. Comics to be hilarious and relatable.  If you’ve ever written a thesis or worked on a project with an advisor who seemed like God, you probably can understand the predicament of our heroes here.  

                Unfortunately, July 2010 was over a year ago so I don’t get back there too often.  I caught up on all new comics since February 2011 a few days ago.  I posted a few of the best ones here.

                If you want to read more: www.phdcomics.com

One. Technicians, graduate students, post docs or just lab-hanger ons are required to travel the cheapest way possible to every conference.  Considering conferences are suchabigdeal, we’re traveling a lot without spending any money.  I’ve worked hard to avoid conferences because I don’t like sharing a hotel room with a stranger for six nights.

 48.1: http://www.phdcomics.com/comics/archive.php?comicid=1425

Two. Truth.  Anything could happen outside and those of us in lab wouldn’t know.  I had friends in graduate school without lab windows so they didn’t even know if it rained!  We had an earthquake here in August and most of the people I worked with thought it was someone moving an especially large piece of lab equipment.  Thank God for Facebook or we’d be completely cut off.

48.2: http://www.phdcomics.com/comics/archive.php?comicid=1427

Three. This happens.  Entirely too frequently.

48.3: http://www.phdcomics.com/comics/archive.php?comicid=1429

Four. I wrote a pre-application for a grant several months ago.  Some of the stuff I needed money for was already finished.  Science is a game.  Sadly, I lose often at it.

 48.4: http://www.phdcomics.com/comics/archive.php?comicid=1431

Five. This year, I got married and needed two whole weeks off.  When do you bring that up?  It was unspoken until about a week before I left and that was only because I had a student starting and it became imperative for me to say “Yeah, I’m not going to be here.  Remember?”  Remember?  Like we’d ever had that talk??  Once in graduate school, I actually emailed my boss from Florida saying I wasn’t going to be in that day or the next.  That was kinda low. 

48.5: http://www.phdcomics.com/comics/archive.php?comicid=1432

Six. This is true for some disciplines and most definitely true for my own.  Oddly, in my post doc, I’ve found people whose advisors read their theses multiple times and gave them edits.  My god.  That’s so …unfortunate.

48.6: http://www.phdcomics.com/comics/archive.php?comicid=1441

Seven. I’m so tired of dressing like a college student.  I’m 31.  I don’t want a red X through my business clothes any more.  If you dress nicely in lab people wonder what’s wrong with you.

48.7: http://www.phdcomics.com/comics/archive.php?comicid=1446

From DNA to Protein, Step 3

Okay, this is it!  The last step!!  As always, review the previous posts in this series if you’d like to understand more of what is going on here.  

Let’s remember what we did in Step Two: Bacteria are the best tool scientists have for making protein.  One little bacterium is not sufficient for making as much protein as we need.  In fact, scientists typically grow liters worth of bacteria.  While the bacteria grows and is churning out its own proteins necessary for survival, it is also making our protein of interest: GST-RED.  Eventually, we collect all the bacteria, which are full of GST-RED, and freeze it until we’re ready to finish this whole thing out!

BACKGROUND/EASY EXPLANATION

                This part is like cooking in that the scientist needs to understand subtlety.  The broad strokes of what I’m going to explain to you will read like a recipe, but any chef knows that grace, control, and keen understanding is necessary to actually make delightful meal.

                With that in mind, let’s get that pellet of E. coli bacteria out of the freezer.  YUM.  In case you are wondering – no, it doesn’t smell good.  After working in a lab of 12 – 18 people (depending on the time of year) and being surrounded by growing bacteria every day, the smell really doesn’t bother me anymore.  However, if you aren’t used to it, it can seem downright disgusting.  

                  The pellet is individual bacterial cells filled with GST-RED.  The first step to getting to the GST-RED is to break open the bacterial cells.  Several ways exist to do this (sonication, cell disruption, French press, lysozyme) so I’m not going to bore you with the details.  Instead, I’m just going to tell you it can be done!  A visual diagram of this step can be found in Figure 46.1.

                Remember, breaking open cells is like bombing the walls of a house.  Everything was nice and organized inside and then becomes a downright disaster.  Everything you don’t want is also mixed up with what you do want.  Following the breaking open of the bacteria, we are left with a great goop of stuff that includes DNA, fats, and all the other bacteria proteins in addition to GST-RED.  How in the world do we separate everything from GST-RED?  HOOOW?

                The first step is through centrifugation.  By spinning this mess at high speeds, anything very heavy is going to straight to the bottom of the tube to form a pellet.  GST-RED is not something that will be found in the pellet, but things like broken membranes and large DNA pieces will be.  Awesome – we’ve gotten rid of some of our problems!   To visually see this step, check out Figure 46.2.

                The second step is to use something called resin.  Resins can be bought at a variety of companies.  They are composed of small, gel-like beads that are linked with a particular compound.  In our situation, our resin will have beads that are connected to a compound called glutathione.  It’s not important what glutathione is beyond the fact that the GST within our GST-RED protein loves to bind to glutathione. 

If we take our centrifuged sample and mix it together with this resin, GST-RED is going to bind to it but everything else should not.  The other proteins do not have GST and have no interest in glutathione.  To see visually see this step, look at Figure 46.3.

Once all the GST-RED is bound to the resin, it is washed a few times to get rid of any other remaining proteins.  Then you are left with the left hand side of Figure 46.4.

The last step is to just add a small amount of elutant to the resin.  Elutant is something that either the resin likes more than your protein or your protein likes more than the resin.  In either case, the protein is kicked off the resin and the scientist collects it in a tube.  If all goes well, you have successfully achieved the final box of Figure 42.1: only red triangles.

Congrats!  We’ve made it!

Because of the nature of this step, there are many many many factors to consider.  I’m going to skip the more complicated explanations of anything because I could be writing all day.  If you have any specific questions, please feel free to ask!

From start to finish, this entire protocol of Steps 1 (preparing DNA), 2 (growing cells) and 3 (purifying protein) takes about two weeks.  Step 1 is usually the longest: it lasts about a week to go from nothing to confirmed plasmid.  Step 2 takes ~ 24 hours.  Step 3 takes anywhere from 24 – 72 hours.  It really depends on what kind of fanciness needs to be done to achieve purified protein.

Do you think you can do it now??

REFERENCES

Me, myself and I

Carbon Monoxide Poisoning

This post was requested several weeks ago.  My apologies in not pulling it together sooner.

Also?  The hemoglobin protein is directly involved in Sickle Cell Disease.  Check out a small blurb on this subject on Mini-Amedeo - LINKY. (http://miniamedeo-amedeo.blogspot.com/2012/01/sickle-cell-trait.html)

**        

                We’ve all heard of it.  We all think we understand what it does.  We’re all convinced it’s red.  Such is the general knowledge of hemoglobin.  Now, I’m going to take you now on a guided tour of this protein.  Come along.

                Figure 47.1 shows you one molecule of hemoglobin.  It’s a small protein (the α subunit for humans is only 142 amino acids, P69905).  You’ll notice, however, that hemoglobin isn’t solely comprised of amino acids!  There is a large, flat molecule associated with it called a heme group (colored red).  

Once the protein has been created by the ribosome (Central Dogma post…), a heme group nestles itself inside the hemoglobin protein molecule.  Heme groups are not protein.  They are not encoded by our DNA.  They are simply molecules that our cells make for the sole purpose of sticking them inside hemoglobin.  Think of lovely wrapped present.  The hemoglobin protein is the wrapped box and the heme group is the bow – tacked on the top, but totally completes the package.

Figure 47.2 shows you exactly what a heme group looks like.  Don’t worry, you only need to understand one thing about the heme group: it binds oxygen.  This means that a hemoglobin protein without a heme group cannot bind oxygen.  It is, in essence, useless.

Hemoglobin is an interesting protein.  

For one, it is a tetramer.  The picture in Figure 47.1 is not complete.  Some proteins are content to hang out on their own but others like to be in groups.  Hemoglobin is one of those proteins.  In fact, it likes to be in groups of four.   This means that the mature hemoglobin proteins in our blood look essentially like the above picture times four.  This is shown more easily in Figure 47.3.  Each individual molecule carries its own heme group so this means that a tetramer of hemoglobin can bind four oxygen molecules total.

Secondly, hemoglobin helps itself bind oxygen.  This property is called cooperativity.  When one heme group within the tetramer binds oxygen, it becomes more likely that the other heme groups in the tetramer will bind oxygen.  It may seem like an odd concept at first, but oxygen is crucial to our survival.  Hemoglobin’s job is to pick up oxygen at our lungs and then carry it to various places in our body.  Anything that will make the pick-up of oxygen more efficient, such as the cooperative nature of oxygen binding, is greatly desired.

The left of Figure 47.4 shows you what oxygen looks like.

The right of Figure 47.4 shows you what carbon monoxide, also called CO, looks like.

My, my.  They look really similar, don’t they?

They actually are really similar.  

Sadly, they have one huge difference.  Oxygen likes binding to the heme group in hemoglobin, but is perfectly content popping off when needed.  Obviously, hemoglobin is meant to drop oxygen off at cells so the oxygen must be able to get off the heme group when necessary.  Carbon monoxide, however, has no interest in getting off.  It loves the heme group and will stay there.  Forever.

This leads to a two-fold problem when a person continues to breathe in carbon monoxide.

One.  All the hemoglobin traveling to the lungs to pick up oxygen are picking up carbon monoxide instead - carbon monoxide that will never get off their heme groups.  The amount of oxygen available to your cells is going to drop rapidly.

Two. Oxygen is important to your cells.  Everyone knows that we breathe oxygen in and carbon dioxide out (plants do the opposite!), but what is its role once inside the body?  I touched on it briefly in the post Conferencesand Cancer Cells, Part 2.  I’ve placed Figure 21.1 below that reviews how the cell gets energy (which is called ATP).  Oxygen is crucial to the last step, called the Electron Transport Chain.  If oxygen is not around, that entire diagram stops running, which means that the cells are now starved ATP.  Without energy, many essential biological processes simply stop and cells begin to die.

                Carbon monoxide is ordorless, colorless, etc…  You can’t see it or smell it and there’s no way to know there’s a problem until it’s far too late.  Some people have carbon monoxide detectors in their homes.  Many know not to stand in a closed garage with a car running.  Be wise about carbon monoxide.

                This entire post reminds me a scene from the movie “The Client.”  Jerome “Romy” Clifford drives out to a deserted area, runs a hose from his exhaust pipe to the window of his car and tries to kill himself.  It would have worked nicely if it wasn’t for the two kids who happen upon him.  Good movie; better book (John Grisham).  Go read it!

Heme group: a special group of molecules that binds to hemoglobin and is responsible for binding oxygen.

Tetramer: Protein molecules sometimes come together to form higher order groupings.  A single, functional protein is called a monomer.  Two protein molecules that come together are called dimers.  Three = trimers.  Four = tetramers.  This goes on as high as you can imagine…

Cooperativity: The act one of process helping another (there’s more to this definition, but let’s leave it at that for now).

REFERENCES

Zumdahl, Steven S. “Chemical Principles, 4^th Edition” (2002) Houghton Mifflin Company, Boston, MA.

Alberts et al. “Molecular Biology of the Cell, 4^th Edition.”  Garland Science, New York, New York. (2002).

Grisham, John.  “The Client” (1993) Bantam Dell, Random House.  New York, New York.

Hemoglobin PDB code: 1HHO, pictures were made in PyMOL