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 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!
I empathize with you; things sound so easy and straight-forward to do until you actually attempt it and unforeseen glitches pop up. Same thing happened with me when I thought finding emails of contacts would be so simple for my survey. I never thought about how difficult it is to catch bacteria--sounds harder than catching fish!
ReplyDeleteDylan, in your posts I get a feel for what you are doing but what are you learning beyond the obvious lab techniques etc? I'm just curious.
ReplyDeleteI am learning a lot about how bacteria function and how plasmids work. It's interesting seeing the more in-depth side of biology from what I learned in AP bio.
DeleteIt's always the little things that end up being more complicated than expected! How did you measure the optical density of the bacteria?
ReplyDeleteI used a photospectrometer. It reads the absorption of light by a solution by shooting light into it.
ReplyDeleteHi Dylan! Getting the right optimal density sounds like quite a challenge! Is human error (timing being a bit off in reality) a big factor in obtaining the optimal density or is it a question of uncertainty about what the timing needs to be, requiring a trial and error approach?
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