MCB 3020C Lecture Notes - Lecture 24: Green Fluorescent Protein, Flow Cytometry, Winogradsky Column
Document Summary
The winogradsky column: provides a variety of microenvironments for phototrophs (anoxygenic and oxygenic), sulfur-reducers, and other microbes, serves as a long-term source of bacteria and archaea for enrichment cultures. Sterile seawater plus minerals supplemented with different nutrients in each wel. Deposit single cells from seawater into individual wells of microtiter plates (obtain individual cells by flow cytometric sorting or dilution). Monitor for microbial growth or specific target genes: wells showing growth contain stimulatory nutrients. Two dyes are used, one that stains live cells and the other that stains dead cells, based on integrity of cell membrane. Green fluorescent protein (gfp) can be genetically engineered into cells to make them autofluorescent. Specific genes can be used as measure of diversity: techniques used in molecular biodiversity studies, dna isolation and sequencing, pcr, restriction enzyme digestion, electrophoresis, molecular cloning. Restriction digest total dna and then shotgun sequence, or sequence directly (without cloning) Single-gene phylogenic tree: phylogenic snapshot of most.
