Week 5- Preparing to Map a Transposon

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. I categorized each mutant as either fast to oxidize and motile, slow to oxidize and motile, delayed motility, revertant motility (irregular shape, picked up an additional mutation that let it swim), or non-motile. I chose two from each category to have a total of 10 mutants for the upcoming experiments. I preformed another optical density test on my flasks of the wild type bacteria growing in the presence of no food and in the presence of lignin. My values implied that there was bacterial growth in both flasks, but I don’t really trust that result. The bacteria should be starving and dying in the flask with no food source. With the size of the biofilms stuck on the glass, I don’t think the amount of cells present is being represented accurately either. I made four different types of media today, which led to a lot of plate pouring. We sure are going through plates quickly! I also inoculated all 28 of my mutants so that I could use them on Tuesday to make freezer stock solutions, and isolate their genomic DNA!

The way I initially labeled my mutants is becoming tedious beyond compare, so the first thing I did Tuesday morning was rename all of my mutants and included the key in my lab notebook. Now it will be much easier to fit their names on the lids of all the tiny containers! With the overnight cultures I prepared Monday afternoon, mixed with some glycerol, I created freezer stock solutions of all my mutants. I am storing them in the -80 degrees Celsius freezer so I can leave them there as long as I want, but will have them whenever I need them again. The exciting event of Tuesday was that I got to isolate the genomic DNA in my 10 selected mutants with the Wizard Genomic DNA Purification Kit! I had never done that before; it was super neat! I saw the cotton-like string of DNA suspended in solution during this process. Isolating the genomic DNA will bring me one step further in the process of identifying the negative regulator on one of the Mn oxidizing genes.

With the battle against biofilms, we are trying yet another approach to our carbon source experiment. Ascorbic acid reduces the insoluble Mn(IV) oxides back into the soluble form of Mn(II). This is helpful because it keeps more of the cells in solution by decreasing the amount of clumping. However, ascorbic acid also kills the bacteria, so we couldn’t just proceed with one container of culture and add ascorbic acid to it every time we wanted to quantify the cell growth. So, I made a stock solution of the lignin containing mixture and distributed it into 8 different tubes. This way all tubes start out the same, I will only use and inject one tube with ascorbic acid at a time; the rest of the tubes will be left growing until the next time I want to measure the growth. It seems like a pretty clever solution, but it does introduce some variables. I plated a series of dilutions of each of the four starting cultures. I learned how to use the NanoDrop instrument today for quantifying the starting amount of the genomic DNA I extracted this week. It’s like the spectrophotometer I’ve been using but cooler because it’s teeny tiny. For the whole process of identifying the negative regulator, I must first map the transposon (which I used to disrupt the gene encoding the negative regulator). The first step of this process is a restriction digest of the genomic DNA, which uses a restriction enzyme to cut up the DNA into a bunch of little pieces.

For the carbon source experiment (the one that will be used with ascorbic acid), I plated a series of dilutions again from the same staring cultures. The dilution plates from Wednesday were incredibly overgrown by Thursday morning which tells me there must have been a contamination since one of the plates is a 100,000 fold dilution… I am worried the media I used for this experiment was contaminated. To quantify bacterial growth in the presence of lignin, I am taking the approach of diluting the culture on plates and counting colonies rather than performing optical density tests like I did last time, since that data seemed pretty unreliable. To continue categorizing my mutants by their phenotypes, I’m growing them up in liquid as well as plates. My mentor has observed differences in Mn oxidation that depended on the substrate, so I will use this phenotype to distinguish between my mutants. Having completed the restriction digest on Wednesday, on Thursday I proceeded with the next step of the process which is “ligation”. On Wednesday I broke up all of the gDNA into little pieces, and on Thursday I turned those pieces into circles with the help of a ligase enzyme. The purpose of this step is to have the transposon bound up into a plasmid for the upcoming transformation experiment.

On Thursday I plated the media I was concerned about being contaminated to see if anything would grow (i.e. if there was any bacteria in there), and the plate was covered in a smear of colonies by Friday morning… Yikes! I had to toss that media away and will use a different batch of that type of media for the rest of my experiments. Thank goodness the carbon source experiment dilutions that I re-did turned out much better this time, and there were much fewer colonies so that I could count them. I counted the colonies on each of the plates and multiplied that value by the dilution factor to determine the colony forming units per mL. It’s amazing to think that millions of colonies are in a single mL. I will keep repeating this diluting, plating and counting process for a week or so. On Friday I diluted and plated another set of the cultures, after adding ascorbic acid to break up some of the oxide clumps. In regards to the other project, I had done a restriction digest with the gDNA, a ligation, and on Friday I did a transformation. The transposon was bound up into a plasmid during the ligation, and on Friday I put that plasmid into E.coli. I did this by heat shocking the E.coli so that its membrane would open up and the ligated gDNA could insert itself inside. I then had to feed the E.coli (gave it some hearty media to sit in) and incubate it at 37 degrees Celsius for an hour because it takes a little bit of time and care for the E.coli to take up the transposon and start expressing the genes. The transposon contains a kanamycin resistant gene, so by plating the E.coli onto kanamycin-containing plates, I can select the colonies that have taken up the transposon because those will be the only ones growing. All of these steps are part of the process of mapping the transposon.