July 16-18, 2013
Finally after over a month of preparing the DNA fragments it was time to analyze the activity of the P5 promoter at each different position in the chromosome. To do this we conducted both a flow cytometry experiment and a fluorimetry experiment.
At the most basic level both of these experiments work by placing the samples of the DNA, that has our desired fragment inserted into the chromosome, into the flow cytometer and the fluorimeter. These machines then analyze the fragments and give us data on: the activity of replication, the absorbance of the cells, the fluorescence of the cells, the copy number of the cells and the concentration of the proteins inside the cells. All of these measurements are tracked over time, and after the experiments we analyze the graphs of each of these variables vs. time.
Of course it is not as simple as just placing sample of bacteria into the machines in the laboratory. Before placing the bacteria into the instruments you have to prepare the samples. To do this you first have to run an upshift experiment. An upshift experiment follows the growth of the different strains of bacteria by measuring the optical density (OD) of the population of cells. The OD is important because it follows the growth of the population of the bacteria. The more cells you have, the more light will be absorbed. A spectrophotometer measures the absorbance of the sample and gives you a value of this absorbance, which is the OD. From these values you can make a growth curve which shows the growth of the population of the bacteria.
I should mention that I was not only measuring the OD of the bacteria with the insert of the P5 promoter. I used three other strains of bacteria. We measured the OD of strains of bacteria with different regulatory elements (rrnBP1, P2, P5 and gyrase) inserted into the chromosomal DNA. These acted as controls because we already knew the results from these other strains of bacteria. From the growth curve of each of these strains of bacteria we could tell when the cells reached the exponential phase. At this point we began to take samples of the cells every 20 minutes. These samples were spun in the centrifuge, and then prepared for the flow cytometer and the fluorimeter.
To prepare the cells we killed the cells with FIX (a chemical to kill bacteria), centrifuged them, added PBS, centrifuged them again and then put them on ice. Just to give you of an idea of how much work this was, we made about 200 samples (over two days) of the different strains. The master student who did these same experiments last year, but without the phage t7 P5 promoter made about 60 samples of her bacteria. Even 60 samples was considered too many, so 200 samples was exhausting. It was a lot of pipetting, centrifuging and measuring.
Then at the end of the day we used these samples for the fluorimeter experiment. First we used a robot to make a 1:10000 dilution of each strain in three different mediums: glucose and two types of casamino acid: CAAO2 and CAAO5. A growth medium is a solution that supports the growth of the bacteria cells. Then we took these dilutions and placed them into the fluorimeter. The machine measured the OD and the fluorescence of the samples throughout the night, and the following day I made graphs of the data. The preparation of the cells took a full day and we repeated this whole process of preparing more cells the next day. We did this so that we would have more samples and thus more data points for the flow cytometer.
Finally the day before Bianca needed to have all the data collected we used the flow cytometer to study the fluorescence and the activity of each individual cell. The flow cytometer was in a fancy, high-security lab downstairs, where another team at LBPA studies the HIV virus. I had to go through multiple doors to make sure that the pressure of the inside lab was kept constant. I also had to wear a smock, air mask, hair net and slippers in the lab! I looked ridiculous, but I needed to use the flow cytometer. Anyways, I spent the entire day passing the 200 samples through the laser of this instrument.
Unfortunately, over half of the samples were useless. The laser did not detect any of the cells in the sample, and so we did not have good results. This could have been because the cells were washed away during the preparation, or it could have been due to some other problem that occurred at some point over the last three days. Maybe the centrifuge was not set to the proper speed, and so the cells did not stick to the bottom of the test tube as they were supposed to, or maybe I was too violent when I put the PB into each test tube. There is no way to know, but we continued with the data that we had from the good samples (the samples that had enough cells to be detected by the laser) and with the data collected from the fluorimeter.
The following day Elisa and I made the graphs from the results of the fluorimeter. There was a lot of inconsistent data, but the overall curve to each graph was consistent with what we had expected from theoretical estimations. We believe that the inconsistent data was due to a malfunction with the fluorimeter, and so Elisa and I went through the data points and corrected the numbers that did not fit the trend. We replaced every number that was un-characteristically low with a number that fit the trend. These low values in the data set occurred at the same place in each set. This was reassuring that it was a problem with the machine, and not a problem with our sample.
After constructing the graphs we sent them to Bianca. She was pleased with our results, and presented them to a group of theoretical biologist at the University of Paris. The data showed that there is a difference in the activity of the rrnBP1 promoter compared to the activity of the phage t7 P5 promoter. I will explain what this means and the conclusions from my experiments in my final lab report, which I am working on this week. I will be sure to post a copy of the report on here so that you can see my graphs and conclusions.
My internship is almost over. This week is the last week, and then I will be reading a book on a beach in Greece, far, far away from my email and the lab. But before I get to run away to a Greek island, I will write a few more posts. I am working on a series of posts. One is on the epistemic values that are inherent to science, another is my lab report, and the final post that will be a summary of my internship focusing on the three themes: epistemology, broader impacts and ID/TD. So more ideas are coming this week!!
A bientôt!
axio