Kai Ng's blog

This week was not as busy as previous weeks. I did not have to run time-points like the previous weeks, and did not grow Pseudomonas putida. Most of what I did was to wrap-up.  I finished the measurements from last week’s experiment, finished analyzing last week’s data, ran an experiment for catalase activity with different concentration of catalase, edited my PowerPoint for the final presentation, and finished up my final paper for this internship.

There’s only one more week left for this internship. Everyone in the lab was so busy. It could be that everyone was wrapping up their experiments. This week I repeated the experiment from last week but with different concentration levels of Iron: 1.8 and 9 uM Fe. The measurements were the same: catalase activity, hydrogen peroxide concentration, the presence of pyoverdine, dissolved and particulate manganese (Mn), manganese oxide (MnO_x), and the culture density.

Pyoverdine is a organic ligand that binds to iron molecules. At a low iron environment, Pseudomonas putida and some other bacteria produce pyoverdine to find iron for uptake. On top of our manganese research, we were also interested in looking at the pyoverdine secretion for this week’s experiment. I grew the P. putida in minimal medium A with 100uM manganese, Mn(II), and different concentration of iron (0.37, 18, and 100uM). There was only four time-points collected for this week.

I started this week by running my last time-point from last week’s experiment which was growing P. putida with and without light in minimal medium A with 100uM manganese and 0.37 uM  iron. Then Matt and I wanted to look at the differences on filtered and unfiltered medium with P. putida. We believe that if the P. putida produced catalase in the medium, we would see the concentration of hydrogen peroxide decrease over time on the time scale we set up: 14, 8, 4, 2, 1, 0 minutes. However, it did not came out as we expected.

This week Matt and I did an experiment with six time-points. We prepared any needed medium for growing Pseudomonas putida. The next day we grew the P. putida in LB medium overnight. The culture was then inoculated into minimal medium A the next morning. We spent the rest of the week running experiments on the P. putida looking at how they would react when 100uM of manganese chloride (MnCl₂) and 0.37uM of iron chloride (FeCl₂) were added.

Last week we found the fluorescence from the medium with Pseudomonas putida was way lower than the fluorescence from our control medium. This meant that the P. putida had changed the chemistry of the medium and was breaking down the hydrogen peroxide(H₂O₂) in the medium. In the presence of H₂O₂,  they produced an enzyme called catalase that would break any H₂O₂, and almost every organisms that are expose to oxygen have this enzyme to prevent accumulation of H₂O₂.

This week was all about hydrogen peroxide(H₂O₂) . The H₂O₂/amplex red protocol, which I have been practicing, was used to measure the H₂O₂ concentration in a minimal medium with bacteria called pseudomonas putida GB1.  We want to test if the bacteria would produce H₂O₂. So far we only have collected six time points. My mentor, Matt and I planned to take another point on this coming Monday.

I have been practicing on building calibrations curves. This is important because the curve is used to calculate the concentration of tested samples. I have no problem with the leucoberbelin blue calibration curve which is used to calculate the concentration of KMnO4. So far the results were all very similar. On the other hand, the hydrogen peroxide (H₂O₂) calibration curve varied in every trial. A lot of noise came out while reading from the spectrophotometer with H₂O₂.

This week, most of my time was spent on practicing with hydrogen peroxide – A-red assay, a couple of Leucoberbelin Blue (LBB) assay to build calibration curves I also worked with Kati Geszvain and Christine Romero. They let me work a little bit with bacteria, such as streaking plates, inoculating bacteria from plates to liquid media, sterilize media and pipette tips in the steam sterilizer and oven.  As I said last week there were some complications in this protocol procedure. My mentor, Matthew Jones, and I had been working on clarifying out the procedure.

This is my first time doing research internship. I’m working with Matthew Jones this summer who is out in the Black Sea for the first two weeks and a half. However I’m not alone. In this situation email come in handy. We email each other for updates and questions. Plus I got a lot of help from all the people who work in the lab. This week  I did a lot of practice with leucoberbelin blue (LBB) protocol to build a calibration curve, and grew some bacteria with help from Christine. Other people in the lab also helped me and showed me where things were placed.