Biology 101...

My transfections failed again.

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[Editor's note]: I had originally wanted to provide an update to my various ongoing sagas in the lab. However, it dawned on me that most people won't have any idea what I'm talking about and thus it won't mean a thing to you. So I've decided to provide a brief explanation along with it. So, here is the first of what will surely be many science-mired posts....

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For those of you who don't understand the meaning behind today's first sentence -- it has to do with gene cloning, which I am currently in the process of doing in my lab. I won't get into why just yet, but be assured it's for a worthy cause, probably. The point is, gene cloning is relatively easy, and yet I have been unsuccessful with this single experiment for the past 8 weeks. Up until now, I have been unable to produce any growth of genetically altered bacteria. After many days of eliminating possible variables, I determined the problem was a particular faulty enzyme (SAP, for those who know). Certain that I had fixed the problem, I tried another transformation (gene-alteration) and discovered to my chagrin that my vector control transfection grew colonies!

(That's not supposed to happen)

Anyway, that's the update. I'll be trying again next week from scratch, and hopefully it'll turn out that today was just a fluke. In the meantime, I'll try to churn out a simplified explanation of cloning, which I'll post later.

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Edit: Here it is:

Gene Cloning (note: boring science stuff follows... read at your own boredom)
Cloning a gene into bacteria is really quite an easy process, albeit a slightly complex principle. So let's start at the top:
One step in control of gene expression is transcription factors. These proteins bind to a particular region of DNA called the promoter. This binding drives expression of that gene.
My goal is to test the ability of a particular protein (Stat5) to bind to the promoter of a particular gene (EBF). The EBF promoter has three potential spots where Stat5 might bind, so the first step in a long line of experiments is to express these sites individually, and see if they respond to Stat5.

First, you make DNA fragments that consist of the section of the promoter you are interested in (i.e. the Stat5 binding sites). This is done by use of a process called the polymerase chain reaction (PCR). Basically, it lets you pick two points on a DNA strand and multiply everything in between. By some clever manipulation, I also stuck two short sequences at the ends of the fragment, called Xho I and Kpn I. I'll explain their importance in a bit.
So, now you have a bunch of little DNA fragments that you want to introduce into a bacterial cell. Luckily, bacteria have a unique ability to take up random DNA that is floating around and incorporate it into their own DNA (this is one reason bacteria can mutate easily). So, all we have to do is let it suck up the DNA and we're done, right? Ah... if only it was that simple...

Bacteria can only express circular DNA (called plasmid DNA), so you need to find a way to make the fragment circular. To do this, you utilize a pre-constructed plasmid. Plasmids are engineered by biotech companies and are full of all kinds useful characteristics. For instance, the plasmid we're going to use has two particular short DNA sequences in it: Xho I and Kpn I. Sound familiar? These are restriction sites and they are recgnized by the restriction enzymes Xho I and Kpn I. These enzymes cut the DNA right down the center of their correlative sites (and only at those sites), opening up the circular plasmid, and leave half of an Xho I site on one end and half of a Kpn I site on the other. You also cut the promoter fragment you made earlier, and they leave the same half-sites on the ends of that.

Due to some boring molecular stuff, you typically treat the plasmid with phosphatase in order to improve the efficiency of the insertion. This is the SAP step at which I originally thought my experiment is going awry, but no matter...
Back to our plasmid. Because you left the ends of your now-linear DNA with different sequences, they should not reconnect to form a new circle. It's like trying to plug your hairdryer into a European outlet. The plug and the socket won't match without some adaptor that fits each. Your promoter fragment is just that adaptor. So you mix the two together with some enzymes that help connect (ligate) the two, the plasmid reforms a circle, and you throw the whole mix in with some bacteria. Heat shock (or electrical current) causes the cells to form pores through which the plasmid (carrying your gene) can pass. And so the bacteria take up the DNA and express it as their very own. You then grow the bacteria on agar plates, pick some clones, and use them for further experiments. Congratulations, you've just cloned the EBF promoter into a cell.

However... how do you know that the bacteria that is growing actually took up the plasmid DNA? Well, the plasmid we use (like most plasmids, actually), contains a gene that confers resistance to the antibiotic ampicillin. If the fragment inserted correctly and caused the plasmid to reform a circle, the bacterium will be able to express this gene, and it will be ampicillin-resistant. If it doesn't insert properly, the circle will not reform, the bacteria will not express the gene, and they won't be resistant. Therefore, all you have to do is grow the cells on agar that contains ampicillin, and the correct cells will grow, while the incorrect cells will not. Easy.

Now, here's a quiz: go back and read what results I got from my experiment today, and see if you can figure out why this is a problem.
10 points, open notes.