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.