To start from the beginning, the Niles Lab here at MIT is all about coming up with new molecular tools to help scientists study malaria. Malaria is caused by a parasite from the genus Plasmodium, and to date, science knows very little about these critters. Not a single drug that is currently used to treat malaria has been rationally designed…they have all been lucky accidents or the result of glorified guess and check. Instead of science understanding the ways in which malaria parasites hijack your body and target these particular pathways, science has only been able to bombard malaria parasites with different chemicals and see what does the trick. The current method is inelegant, expensive, and doesn’t really provide the suite of medicines we need to eradicate or even treat malarial infections.
Thus, our lab is most interested in how we can increase the general knowledge about malaria parasites: both the pathology and how it makes people sick, but also more about the basic biology of the parasite. To do this, we want to find out more about their genes and proteins. Basically, when we think about how a cell or person becomes who they are, it’s because of their genes, which are encoded by their DNA. DNA is transcribed into an RNA message, which is then translated to become a functioning protein. So---differences in DNA and genes, like the ones that encode for skin color or hair color (or insert your favorite characteristic here), manifest in the proteins that are translated.
There are many similar proteins and genes between different species, and science calls these “homologous” genes. In Greek, homo means similar, and logos means study of---so homology is a study of things that are similar--it has a wide range of other disciplines where this sort of word describes a lot of different things. But in biology, homology looks at those genes or bone shapes or molecules that are the same between different species. The genome of the malaria parasite has been sequenced, but to everyone’s surprise, there were far fewer homologous genes than are usually seen between a newly-discovered organism and other organisms whose genome has already been sequenced. All in all, we don’t really know much of what is going on, and what we know isn’t well understood. Not a good place to be when you’d like to design better drugs, that’s for sure. So, all the members of the Niles lab are working on ways to help science better understand malaria.
For a brief interlude, a history lesson. Clone comes from the Greek word klon, meaning twig. It dates back to the science of creating new hybrids of plants by literally tying twigs from one tree to an existing tree and seeing what happens...if it's successful, the plant blossoms, you can get cross-fertilization and end up with a new plant. Even though this has been going on for millennia, it is only lately that scientists have started to clone on the molecular level.
Molecules are bits of atoms that are somehow bonded together. In this case, cloning on the molecular level means changing the DNA. Again, no crazy half-baby, half-lizard monstrosities here...we're talking about changing just a few DNA bases of the thousands that code for a specific gene that codes for a specific protein. But this isn't as easy as it sounds. Not by a long shot.
Science uses a suite of tools to help us change the DNA in a very specific and targeted manner in order for us to test for our desired outcome. We are, in effect, engineering the DNA (this is where the word genetic engineering comes in, by the way).
My current cloning troubles have dealt with restriction endonucleases, commonly called restriction enzymes in lab-speak. An enzyme is a protein that decreases the activation energy of a reaction. It does this by optimizing the way in which two molecules meet, or by providing a catalyst, or through many other means. Basically, it makes a reaction go forward. Restriction endonucleases are called such because they were originally found in bacteria and were able to selectively destroy foreign DNA so DNA destruction was restricted to that unwanted DNA. Endo means inside, and nuclease means that it cuts up DNA, so we have these enzymes that are able to cut a chunk of DNA. Cool, right?
[This is EcoRI (blue, red, and white) bound to DNA (lavender and light green) - from the Molecule of the Month portion of the Protein Databank]
[The DNA after the restriction endonucleas has cut it in half. See the excess bit of purple and green? Those are the sticky ends. If you have two matching sticky ends, you can effectively add in more DNA to the DNA sequence]Even cooler is that each different restriction enzyme has a different recognition site, so they cut in a very specific place. For example, EcoRI (said eco (like the eco in ecology) RRR one; it tells you that the enzyme is from E. coli---yes, the kind in your stomach---the strain of E. coli, and the order in which it was discovered in the bacterium)
Very cool. Notice, also, that the recognition sequence is a palindrome (GAATTC on top, and on the bottom starting from the left, it's GAATTC as well).
Unfortunately, when you learn all this stuff the first time, no one tells you that efficiency is often not so good. Or the enzyme itself isn't very specific, and tends to cut other sites on the DNA that it is not supposed to. Bummer.
What I was attempting to clone was a new gene that had a portion of about 30 base pairs added in, and in this case, there was one cut I was making that just wasn't working out very well. But---finally. FINALLY. I have my clone. And now I can actually do some studies on this particular clone. Beautiful.
[also---it is Friday. Hallelujah. A weekend of blueberry and raspberry picking, candlepin bowling, teaching, and a concert at Jamaica Pond. Love it!]
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