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

Tryptophan

               I love turkey.  I love cranberry sauce that has the ridges from its can.  Most of all, I love stuffing.  Thanksgiving is a great food holiday!

                I bet we’ve all seen family members taking a Thanksgiving snooze after eating too much turkey.  (I’m sure Uncle Dave’s impromptu nap has nothing to do with the number of beers drunk while lying on the couch watching football…)  Many people feel that sleepiness following large Thanksgiving meals is due to the tryptophan found in the turkey.  A quick PubMed search of “tryptophan and sleep” will turn up 648 results.  Tryptophan does play a role in sleep.  However, tryptophan isn’t only found in turkey – it’s in egg, soybeans, cheddar cheese, chicken, beef, salmon and even bananas!  According to Snopes.com, you will not eat enough tryptophan on Thanksgiving to feel any appreciable effects, much like every other day of your life when you are ingesting many other foods that contain the molecule.  Also, most experts agree that the post-dinner fatigue is probably due to both an increase in metabolism and blood flow to your gastrointestinal tract.

                So… what is this little molecule?  What does it look like?  What else do we know about it?

                Figure 45.1 shows you what tryptophan looks like.  It’s a small molecule of carbon, nitrogen, hydrogen, and oxygen.  Most things in your body are made of the same four elements (although sometimes we throw in sulfur, phosphorous or a metal for fun).  You’ll notice that the left end of the molecule has two rings of atoms.  These rings are cool because it allows tryptophan to be fluorescent!  If you shine light of 280 nm on tryptophan, it will spit light back at you ranging from 300 – 350 nm.  

                Tryptophan is also something that you’ve heard me talk about in these posts before: it is an amino acid.  In fact, tryptophan is an essential amino acid.  What does that mean?  It means our body has no way of making it, so we must bring it into our bodies via our diets.  Our body needs amino acids to create proteins (oh, that pesky Central Dogma post just never goes away, does it?).  Of the 20 amino acid used to build proteins, 9 are essential.  We have the tools (enzymes, precursors) present in our cells to make all the non-essential amino acids, but essential ones must be ingested.  They are “essential” to our diet, if you will.  Without these amino acids, our bodies won’t make proteins properly and, subsequently, won’t run properly.  Do you remember Jurassic Park?  The dinosaurs were all unable to synthesize lysine (another amino acid).  Unless the animals’ diets were supplemented with lysine, they would die.  

                Interestingly, our bodies do have the tools to take tryptophan and turn it into other useful things.  For example, tryptophan will bind to the enzyme tryptophan hydroxylase and out will pop serotonin.  Serotonin is a neurotransmitter that is commonly thought to “make people feel happy.” 

                Serotonin can then, in turn, bind to N-acetyltransferase and be converted into another molecule that will bind to 5-hydroxyindole-o-methyltransferase and become melatonin.  This molecule is involved in our circadian rhythms.

                I will come back to tryptophan in the last post of the “From DNA to Protein” series, which will be posted soon.

                Until then...

Essential amino acid: Those amino acids that must be provided by our diets

Non-essential amino acid: those amino acids that our body can make

REFERENCES

http://www.snopes.com/food/ingredient/turkey.asp

Schaechter, JD and Wurtmann, RJ.  “Serotonin release varies with brain tryptophan levels.” (1990) Brain Research 532 (1-2) pgs 203 – 210

Wurtman, RJ and Anton-Tay, F. “The mammalian pineal as a neuroendocrine transducer.” Recent Prog. Horm. Res. 25, pgs 493 – 522.

From DNA to Protein, Step 2

It’s time to move on to Step Two!  (Again, if you want more background on this post, see the earlier posts in this series and/or The Central Dogma!)

Let’s remember what we did in Step One:  Proteins are strings of amino acids.  Cells are much better at making proteins than any kind of human techniques available.  Scientists use bacteria to make the protein they want.  Bacteria need the instructions to make protein.  Instructions come in the form of DNA.  The end of Step One was a circular piece of DNA, called a plasmid, which contained the DNA that encoded the protein we wanted.  We put that little piece of DNA into a couple of bacteria.

Now what?

BACKGROUND/EASY EXPLANATION

                One small bacterium is not enough to make all the protein we need. 

                To offer some perspective:

                Let’s say you need 50 ug (Microscopes and Photography post) of the protein GST-RED to perform one experiment.

                One protein molecule of GST-RED weighs ~ 0.00000000000000498 ug.  For real.  See how small that is!?  Even if that one little bacterium pumped out 1000 protein molecules, you’d only get ~ 0.00000000000498 ug.  (You are free to count my zeros.  All I really want you to take away from this is how little a protein weighs versus how much is needed to do experiments.)

                How do you make a lot of protein then?  Well, you make a lot of bacteria!

                E. coli bacteria are quite happy to grow in a liquid called lysogeny broth (also known as Luria Broth or simply “LB,” among other things).  It’s a pale yellow liquid chock full of food that bacteria love.  It comes in a powdered form (or you can make it from scratch with laboratory supplies).  You weigh out the proper amount, add water and sterilize it.  The sterilization kills any bacteria that may be living in the liquid or happen upon the liquid before you’re ready for it.  This ensures that once you add your E. coli bacteria that are bearing your plasmid DNA, all the bacteria that will subsequently grow in the LB will also be E. coli bearing your plasmid DNA.

                How much LB do we usually make?  It depends.  It can be as small 1 liter or as large as 24 liters.  Once, I made 85 liters.

               Again, perspective:  For some proteins, 1 liter of bacteria will give you 10000 ug of protein.  See why growing more bacteria is so useful?

                Let’s be nice and say you only need 1 liter.  You have your 1 liter flask of LB.  To it, you add E. coli that has your plasmid DNA.  Then, you place the flask in an incubator at 37°C (98°F) and gently shake it.  The shaking helps the bacteria grow.  

                And you wait. 

The bacteria are so happy with all the food around that they start to multiply.  Usually, they can double their numbers in about twenty minutes.  

As more and more bacteria start to occupy the flask, the once clear yellow liquid starts to become very cloudy.  Very quickly, we have millions of bacteria in a flask that are churning out our protein.  All those cells are jam-packed with GST-RED.  

Like our refrigerators, only a finite amount of food can exist in a particular area.  We don’t want to overgrow the bacteria to the point where they run out of food and start to die.  For this reason, we stop growing the bacteria at a certain point by removing them from the warm temperature and centrifuging (Tales From the Bench post) the liquid + bacteria mixture.  Centrifuging serves to separate the bacteria from the LB.  We are left with a pellet of bacteria that goes in the freezer until Step 3!

If you now refer back to Figure 42.1, you’ll see that I depicted the end of Step 2 as individual bacteria molecules (rounded rectangles) that are full of our protein (red triangle!)

Step Three, the last step!, is how we get it out of the bacteria!

MORE INFORMATION / INTERMEDIATE AND DIFFICULT EXPLANATIONS

Controlling protein expression.  The plasmid DNA scientists use is actually quite sophisticated.  For DNA to make protein, a molecule called RNA polymerase must bind to the DNA ahead of the gene.  When you only have a few bacteria, it’s not necessary to start making protein.  Sometimes the protein you want to make is toxic to the cells or adversely affects their growth.  It is to the scientists benefit to first grow all the bacteria they need and then have them make protein for a short time.  The plasmids allow us to do this because a protein actually binds to the DNA where the RNA polymerase needs to bind.  It blocks the gene from being read.  Once we have enough bacteria, we add a chemical to the bacteria that removes the blocking protein and allows RNA polymerase to bind and protein to be made.

Ensuring plasmid DNA is in all the bacteria.  In the last post, I alluded to the fact that bacteria can spit out plasmids if they have no use for them.  This isn’t good for scientists.  We need the plasmid DNA to stay in there so we get protein.  Again, the plasmids are sophisticated.  Another gene exists in the plasmid that encodes a protein involved in antibiotic resistance.  This gene is never blocked like our protein gene is.  If the plasmid is in there, the bacteria are making this protein which renders the bacteria immune to a particular antibiotic.  In our LB, we put a small amount of antibiotic.  This serves to kills all bacteria except those bearing the plasmid.

                Some finer points exist in this step, but they are minor.  If you’d like to know how scientists judge how cloudy their LB is (although we can, many of us don’t simply use our eyes) or what exactly the chemicals/antibiotics we use are, just ask!

REFERENCES

Me, myself, and I.

From DNA to Protein, Step 1

                 Okay!  We’re going to do it!  We’re going to make a lot of protein and then purify it.  Are you ready?

                Let’s start with a few clarifications first.

One.  I highly recommend reviewing The Central Dogma post before diving into this.

Two.  The protein we are going to make is called RED. 

Three.  To simplify purification, we’re going to attach another protein to RED called GST.

Four. If you don’t follow this step, don’t be dissuaded from reading Steps 2 or 3.  I’m going to try and make each step stand on its own.  Much like a TV show that has an overarching story through several episodes, each episode can also be watched independently.  I’m going to try and make each post in this series be its own story.  If you see how all the pieces fit together – awesome!  If don’t, just read each post for the story it tells.

                This first step is called Cloning/DNA Preparation.

BACKGROUND/EASY EXPLANATION

                Let’s begin with a bit of background about proteins.  The Central Dogma post explains that proteins are strings of amino acids.  We have twenty amino acids in our body and they can be hooked together in any order and to any length.  A protein is a unique sequence of these amino acids.  A protein can be as short as tens of amino acids or as long as hundreds.  The key to making a protein is knowing its exact amino acid sequence.  

                I’m sure most people realize that chemists hook together and/or manipulate molecules.  It would seem to an outside observer of science that chemists would have come up with an easy way to link amino acids together in any order they want both quickly and easily.  If we know the amino sequence, we can just have fancy science hook them together the way we want, yes?

                The answer is no.  Nothing is more equipped to make proteins than cells.  Cells have all the necessary machinery to create proteins quickly and efficiently.  What takes chemists hours to do, a cell can do in mere seconds.  For this reason, if scientists want a particular protein, then we ask cells to make it.  We ask kindly and, in particular, we ask E. coli bacteria* to do it.

                Why bacteria?  They grow quickly (they double their numbers in ~ 20 minutes), we know exactly what temperature (37°C or 98°F for E. coli) they like and we know exactly what they like to eat.  In short, bacteria are easy to grow in large numbers in a laboratory.

The Central Dogma post reminds us that DNA leads to RNA which leads to protein.  If we want bacteria to make our protein, then we need to provide it with the instructions on how to do it.  The instructions are, of course, the DNA that encodes the protein.  

                Bacteria have their own genome (their own DNA instructions) and we can’t fiddle with that without messing up the bacteria.  Lucky for us, bacteria also have very small pieces of DNA hanging around inside them that are separate from their huge genome.  These little pieces of DNA are called plasmids.  Messing with them usually doesn’t mess up the bacteria!  So, if we can put the DNA that encodes our protein inside a plasmid and put that plasmid inside a bacterium, then it will know exactly how to make our protein.

                This step is called Cloning/DNA preparation because it is the step where we prepare a plasmid that has the DNA that encodes are our protein’s amino acid sequence.  For the purposes of this example, our plasmid will encode the protein GST-RED.  If we look at our picture from Figure 42.1, the result of step 1 is a circular piece of DNA.  The DNA that encodes for GST-RED is colored red and the remaining plasmid DNA is black.  

                Once we make our plasmid, we put it inside bacteria and then grow a lot of bacteria that have that plasmid.   This is moving into Step 2, though!

MORE INFORMATION/ INTERMEDIATE AND DIFFICULT EXPLANATIONS

Where do we get DNA?  In the Extreme Solution post, I discussed a technique called PCR.  If we can get a small amount of DNA that encodes the protein we want, then we can make a lot of that piece of DNA with PCR.  We get the initial bit of DNA from whatever organism makes the protein to begin with.  For example, if the protein we want to make comes from humans, we get human DNA.  From here, we use PCR to amplify the bit we want.  We have to amplify it before we can put it inside the plasmid DNA.  If you'd like to know more about this part, just ask, but I'm going to leave it at this for now.

Where do you get plasmid DNA?  Companies like Invitrogen and NEB make plasmids that bacteria will readily take up.  It’s just up to the scientists to insert the DNA they want into the plasmid.

How do you get the plasmid DNA into the bacteria?  The process is called transformation.  We take a small amount of bacterial cells and add in a bit of plasmid DNA.  The bacterial cells have been treated such that their outsides are sticky.  The plasmid DNA sticks to the outside of the bacteria.  We then drop the tube of bacteria into water at 42°C.  The heat causes the bacteria to swell and open up.  The plasmids that were sticking to the outside can then get inside.  After a short period of time (45 seconds to 1.5 minutes), we place the tube of bacteria back on ice.  This closes up the bacteria again so the plasmid DNA cannot get out.  Ta-da!  We have plasmid DNA now inside bacteria.

More questions.  No, I have not addressed every point in this process.  You can ask how scientists insert the DNA they want into plasmids, how we know we’ve done it correctly, and how we ensure that the bacteria keep the plasmids inside them (they have been known to spit it out).  These are all valid questions and if you are interested, I’ll give you the answers!  Just ask!

* - the easiest organism to ask to make protein is bacteria and that is what I'll use as my example here.  We can also ask mammalian cells or insect cells to make protein, as well.  The steps are overall the same but more involved.  

REFERENCES

Me, myself, and I

               

               

               

From DNA to Protein, Introduction

                 Many of my previous posts have discussed work with individual proteins.  In the case of Diabetes mellitus, the importance of insulin was paramount.  In the Spanish Influenza series, I focused heavily on the protein hemaglutinin.  Thiaminase I took center stage in my mini-series on Australian vitamins while Taq polymerase was key to the polymerase chain reaction in An Extreme Solution.

                How do we know these things about individual proteins?  Well, they were isolated from the rest of cells and studied individually.  

                How do you go about isolating a one protein from the soup of proteins available within the cell?  Ah.  This is what my next few posts are going to focus on.  These steps are commonly referred to protein expression and purification.  What I plan to cover is the most straightforward way to isolate protein, but should not be considered the only way to do so or even to study individual proteins.  Scientists have all sorts of tricks in their bags!

                Protein expression and purification is broken into three steps (Figure 42.1):

Step One: Cloning/DNA preparation.  The necessary DNA is represented by the circle.

Step Two: Bacterial growth/expression.  The bacteria (gray boxes) with our protein of interest inside (red triangles) are shown.

Three: Protein Purification.  Eventually we just want our protein (red triangles) away from everything else.

                In Figure 42.1, I’ve given you a pictorial overview of where we are going.  It’s not supposed to make sense right now.  However, I hope at the end of this series, it will make a lot of sense!

                Several layers of understanding exist for explaining the above steps.  Obviously I understand down to the extreme nitty-gritty, which is probably way more than any of you would like to know.  It is also quite easy to get lost in the details and confuse everyone with what I’m discussing.  For these reasons, I’m planning on breaking each post into two parts: the first part will fall under the EASY category and discuss very plainly what is going on; the second part will be more INTERMEDIATE/DIFFICULT and will discuss the same ideas as the first part, but in more detail and with more scientific considerations in mind.  Whether you read one or both parts is completely up to you!  I also won’t dive all the way down to the all minutiae so ask any questions you wish!