Blogs

This week I ran tests on 2,4-dinitrotoluene or DNT, the precursor to TNT. The only difference is that TNT has one more nitro group in the 6 position. Ali and I decided that this would be a good compound to test because it is very similar to TNT, and since TNT behaved kind of weirdly in the data that I have already collected, we wanted to see if a similar compound would do the same thing. As it turns out, the DNT did not behave in the same way as the TNT!

I completely forgot about this week's blog entry! I can't believe it's already Tuesday and I'm just now writing Week 6's blog. My apologies for the belated update. 

I started out the week running samples on the HPLC from the T=0 (where T is time) sediment sample that I'd prepped last week. I ran samples all of Monday.

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.

Monday, Tuesday: I looked into A Field Guide to Genetic Programming.

Wednesday: I continued looking into A Field Guide to Genetic Programming, had meeting with Charles about looking into ephemeral constants that can generate float values.

Thursday: Continued looking into A Field Guide to Genetic Programming, had meeting with Charles about looking into ephemeral constants that can generate float values.

Although this was a shorter week for me as I had no work on Friday, I was still plenty busy.  After my successful PCRs last week, Pete and I selected a few samples that we would clone and clean-up for sequencing to see what was really being amplified in our samples.  Normally, this is a pretty easy process that just involves inserting the selected gene into "host" cells and then growing them on agar and picking colonies that grew successfully with said insert.

I can’t believe I’m already half way through my internship… It has gone by incredibly fast! On Monday I checked on my second round of motility plates and discovered that my mutants varied in their motility phenotype. This tells me that in all of my mutants, although the transposon suppressed the original mutation of the bacteria by restoring Mn oxidation, the transposon was inserted in different parts of the genome. I categorized my 28 mutants by Mn oxidation and motility phenotypes.

One of my main objectives this week was to figure out if the new primers work. There are three new sets of primers - one for bacteria, one for archaea, and one for fungus. It was easy to test the bacteria primers, because there are plenty of bacterial DNA samples in the lab. Unfortunately, however, that was not the case for the fungus and achaea primers. I tried using a couple of samples that might contain fungal or archael DNA, but I got strange results, which were difficult to interpret without definite positive controls.

After one final blank test with the 353-487 primers in which I finally saw no contamination, I was able to use the primers on our archaeal sediment samples and the results were great (Figure below). All the samples showed good amplification and the data we were able to get showed very interesting graph of the abundance of the amoA gene for the sites view graph

This week has been a busy I didn’t get to do many cell counts this time around since most of my time was spent with other things. Since some people in my lab are preparing for a cruise on the 19^th I have been helping out by washing bottles that they will need to take with them, and by Friday all of them where washed and ready. I also spent all of Thursday filtering 800 ml’s of sea water to help out in the lab as well.

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.