Week 2: Peptide Capture 6

     On Monday, I had my first experience with the method I'll be working with this summer aimed at capturing manganese oxidizers from ETM samples with specific peptide binding. One sample uses a specific peptide that someone else found to bind to manganese oxides, another sample is a random mix of peptides as a control for nonspecific binding, and a third sample is just water as further control. This time, peptide capture 6 (PC6), we experimented with the synthetic manganese oxides that we made last week. Prior to the start of my internship, this method hadn't been working for Roberto, so he wanted to get it to work with synthetic oxides rather than using up any more ETM samples if it failed.

     The peptide capture process involves peptides that have a biotin molecule at the end of their amino acid chain. Biotin has an affinity for streptavidin, allowing the peptides to bind to streptavidin-coated magnetic beads. This allows the samples to be washed by holding them on a magnet, removing the supernatant, and resuspending them in the same volume of buffer several times.

    The beads and peptides are mixed and washed a few times, and then the manganese oxides are mixed in and this is washed about ten more times. Ideally, the manganese oxides (MnOx) will only bind to the peptides in the first sample, although a few may nonspecifically bind to the random mix as well. We used the LBB assay to see if the samples contained MnOx after washing. Unfortunately, all three samples tested positive for MnOx, indicating that the experiment had failed. Further examination under the microscope showed that none of the samples had any consisted MnOx binding, but that all binding was random and scarce and approximately equal between samples.

     On Tuesday, I checked the Bascillus-SG1 culture that I inoculated the Friday before. The turbidity had greatly decreased, there was a clump of something at the bottom of the tube, and there was an orange/brown ring (oxides) around the tube at the surface of the media. What had happened was that the Bacillus had sporulated as a result of nutrient loss in the media, decreasing turbidity, and sank to the bottom, leaving the clump. After sporulating, Bacillus begin to oxidize Mn, hence the ring of oxides. I also got to make Lept and K media on Tuesday, which was mostly just mixing things, and the following day I poured them into plates.

     For much of the week, Roberto was writing his quarterly report for Dr. Tebo, so I did a lot of article reading. Among the topics I read up on were the bacteria isolated from underwater basalts at Loihi Seamount. Apparently one of Roberto's next projects, which I may have a chance to work on, is to try to grow one of these bacteria -- a Halomonas -- in vitro. What is particularly intriguing about these bacteria is the potential that they may alternate between lithotrophic and heterotrophic modes of metabolism. On a somewhat unrelated note, I also read an article about using biogenic manganese oxides to oxidize diclofenac in sewage treatment pant effluent water, a cheaper and more effective method than ozonation and chemical manganese oxides.