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P. putida with 100uM Manganese - Week 6

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.

On Monday, I prepared some minimal medium A, three 1L and one 0.5L, and autoclaved them. After I made the medium, I did a LBB calibration curve with KMnO4 in distilled water with concentrations of 0, 1, 2, 3, 4, 5, 6, 8, 10, 12, and 16uM and one in minimal medium A with concentrations of 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, and 5uM. The calibration curve in distilled water had a better linear fit than the one in minimal medium. The concentration I used in the minimal medium curve was probably too low. I would repeat the calibration curve in minimal medium again at a higher concentration. I did a calibration curve for formadoxime assay in 0.1% hydroxylamine hydrochloride (NH2OH ∙ HCl). All these calibration curves could be used to calculate the concentration of a solution by measuring the absorbance.

I grew some P. putida in LB medium on Tuesday. I expected the P. putida to have grown on Monday afternoon, but it did not. I was hoping I would have the culture ready by the end of the day. After about 9 hours, the LB medium was still clear. This indicates the P. putida was still in lag phase after 9 hours. And while I was waiting for the P. putida to grow, I made a hydrogen peroxide calibration curve with 0.05% phosphate buffer and a formaldoxime calibration curve in minimal medium A.

On Wednesday, I added glucose, Hepes, and trace element metals into the minimal medium A I made on Monday. These chemicals were added in order to keep the bacteria alive.  Then we added 100uM of Mn and 0.37uM of Fe in each of the six 500mL minimal medium used to grow the bacteria. Three were exposed to light and three were wrapped with aluminum foil to prevent exposure to light. Later on, we wanted to measure the total Mn present in medium by using formaldoxime assay. Next, we wanted to measure manganese oxides that the bacteria had oxidized by using LBB assay. Finally, we wanted to measure H2O2 that could be a product from Mn redox reaction. The data did not fluctuate much in the first three time-points. It made sense because it needed more than 9 hours for the bacteria to grow properly.

On Thursday morning, we observed a yellowish-green color in A +light and C +light. A brownish color was observed in A –light and C –light. B +light and B –light did not change color. I might have done something wrong. We believe the yellowish-green color came from an organic ligand called siderophore. In presence of Fe, P. putida produced this ligand to complex with Fe and try to scavenge the Fe. As it compound with Fe, it could also have bound to Mn and scavenge Mn as well. About 2 hours later, the yellowish-green color turned into a brownish color due to the presence of MnOx. This meant the bacteria had oxidized MnCl2 to MnOx.

On Friday I did another two time-points, and I noticed an increase in H2O2 concentration. It could be that the bacteria were oxidizing MnCl2 to Mn III.

The coming Monday, we will finish off the experiment with one last time-point. We will also organize and sort out our data next week because we had been really busy with the time-points.