Optical density, induction, and gels, oh my!

Welcome back everyone! I learned a ton of new techniques in the lab this week all from knowing how densely populated bacterial solution are to running a SDS-PAGE of which I will go into more detail later.

The goal for this week was to do pretty much the same thing I did last week, making more pfu plasmid and running a gel on said plasmid, but since those BL21s had grown, I could start determining if the BL21s are capable of producing pfu polymerase. Seems pretty easy right? Grow some more of bacteria, lyse them, and see if they have the protein. How hard could that be? First off, with growing the bacteria, I needed to catch the bacteria at a specific optical density, in other words, the concentration of bacteria in the solution. This proved to be very challenging because bacteria, especially E. Coli., have logarithmic growth. This means that the bacteria grow their population kinda like a log-graph with three phases. The first is the lag phase, where the population slowly increases at higher and higher rates. The next phase is the logarithmic phase where the bacteria exponential grow till it reaches the final phase called the stationary phase, where the population reaches a plateau at the carrying capacity of the environment.

The problem with trying to find the right O.D. (optical density) is that the O.D.s that I was trying to measure were in that log phase. Needless to say, I way overshot some of the O.D.s that I was trying to measure. All was not lost though as I did have a couple of samples that did have an O.D. relatively close to one of my points.

The whole point of me trying to get samples of bacteria at specific O.D.s was to test the efficiency of induction at those different densities. Now what is induction you ask? Well, induction is a method in which to get a cell to produce specific proteins by targeting an operating site on DNA, in my case, the area that produces pfu. There are a variety of ways to induce bacteria, but I only used a reagent called Isopropyl β-D-1-thiogalactopyranoside, which everyone calls IPTG because that name is painful. Essentially, I just add a bit of IPTG to the flasks I was growing my bacteria in and let them incubate for a while. Of course we will not know if it worked unless we do something else to it, and that would be a new type of gel.

A SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, functions very similarly to DNA gel electrophoresis in that smaller molecules travel further in the gel telling us the relative size of the molecule. These are much more of a pain to set up because this gel run standing, sandwiched between two plates, making it difficult to insert the actual gel in between the plates as well as the samples themselves. SDS-PAGEs also require two different gel types, stacking and separating. The stacking gel goes on top of the separating gel in order to make sure the protein bands that we are trying to measure hit the separating gel at the same time. The separating gel, well, separates the proteins based on size. After running the gel, we have to stain it, which I was not able to do quite yet, but I will be able to do it next week, so you all will have to wait until then!

Successes

Hello everyone! I just got back from Chicago in time to update my blog for this week. I wasn't able to do too much being out of state but I was able to obtain some results. First off, that pesky BL-21 that refused to grow the first few times final decided to grow.

Two different batches of BL-21 were used, both worked.

The problem may have come from the BL-21 that I was using, as I used a new batch of cells for this transformation. Next week, I will be working to grow more of the BL-21 and purify the pfu that it is producing.

I was also able to extract the plasmid that I had used originally on the DH5α cells and run a gel electrophoresis on it.

Two separate batches of plasmid were used here with a marker in the middle.

To perform the gel, I had to mix the plasmid with restriction enzymes, a protein which cleaves DNA at specific sites. This is called a restriction digest. From the picture above, each row shows a different digestions or non-digestions with a marker in the middle to show us the length of DNA segments. The bands that are furthest behind are the undigested controls, the two below that are only digested by a single enzyme, while the last ones are digested by both enzymes, and thus have 2 bands of varying lengths. With this, I can determine what plasmid I used because we were not entirely sure to begin with.

Stay tuned in for next week, where I will attempt to purify the pfu from the BL-21!

The Journey Begins!

Welcome to the first "real" post where I tell you guys about what is going on in the lab! First things first, I had to complete a pretty rigorous course on biosafety. Who would have thought that you should not eat or drink in a lab full of pathogens and caustic chemicals? In all seriousness though, I was finally able to learn the difference between all the different levels of biosafety. These levels range from one to four; one being practically harmless and difficult to contract and four being lethal and highly infectious. For reference, the lab that I am working in is BSL-2, as their are some infectious agents which are not particularly dangerous. Each level also requires different levels of protective measures. For BSL-2, I need to wear gloves, a lab coat, and sometimes eye and mouth protection, depending on what I am working with. For now though, I have not really entered that level as I am just working with E. Coli.

On to my actual experiment, I have been attempting to transform two different types of E.coli, DH5α and BL21. Transforming a bacteria is not what you think it is (though changing how it looks would be pretty neat). All it is is just making that bacteria absorb DNA and express it, in my case a plasmid coding for pfu DNA polymerase, another type thermostable DNA polymerase, and ampicillin resistance. By giving these bacteria a resistance to ampicillin, then they can survive in an LB agar containing ampicillin, which weeds out the bacteria that did not transform. After one failed attempt though, I managed to get colonies to grow with the DH5α E. Coli., but not the BL21 unfortunately.

If you look closely, you can see little, white dots all over the DH5α plate.

Next week, I will be trying to extract the pfu from the DH5α and purify it as well as figuring out why the BL21 would not grow. Stay tuned for more updates!