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

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