Transposon Mutagenesis Read pages 196-200 on the Winogradsky column before the next lecture o Know Winogradsky column

n chart for the final exam Donor Strain (E.coli) o Mutant that cannot synthesize DAP (diaminopimelic acid) Amino acid analog that can make the cell wall Add DAP to medium Maintains the plasmid because it has the Pir gene on the chromosome Means that the pi protein is on the plasmid Has a resistance gene (kanamycin) Recipient strain (Salmonella) o Can synthesis DAP o Cannot replicate the plasmid (because no Pir gene) o Susceptible to Kanamycin Mating o Need to keep both strains happy, so medium should be: DAP +, kan After Mating, you have: o E.coli unchanged (note, transposon can jump in the E.coli) o Salmonella unchanged o Salmonella that has received the plasmid Selection/Counterselection o Selection: to kill the recipient cells that did not mate/no plasmid Add kanamycin o Counterselection: prevents the growth of the E.coli Plate it on medium that has no DAP o Exconjugant Any cell that has been involved in the mating o Transconjugant Salmonella with plasmid Once plasmid is on the Salmonella, o The plasmid gets lost once the transposon jumps (then cell is called transposant) (once the cell tries to divide) Making Yogurt To able to store dairy for long periods of time Is preserved through fermentation produces acid, which makes it hard for organisms who produce spoilage to live o Streptococcus thermophilus

o Lactobacillus bulgaricus (from Bulgaria, Eastern Europe?), acid-tolerant o Lactobacillus acidophilus All three of these bacteria are homolactic acid What are the ingredients in the milk that are important for this process? o Primary protein Casein o Primary sugar is lactose Process o Scald the milk kill the bad germs (pasteurization) o Cool it down to 46-48 C o Inoculate it with yogurt (contains live cultures) o Then you incubate at 46-48 C for 2-3 hours Thermophilis doing most of the work Can transport lactose with in the cell Lactose is broken down by B-galactosidase to produce galactose or glucose Glycolysis occurs pyruvatefermentationlactic acid o Causes pH to drop below 4.6 o Casein denatures at low pH Calcium caseinate begins to denature and separate from the calcium and it curdles, forming a soft white precipitate Soft white precipitate (precipitated casein) curds Whey liquid (like when it separates from the yogurt) In cheese, that is the curds o After incubation, you leave it at room temperature (chill it) S. thermophilis ceases to be active because it doesnt metabolize well at room temp and lactobacilli become active (mesophilic, acidophiles) Lactobacilli do more fermentation, but they have minor metabolic pathways that create more taste and aroma of yogurt o Put in refrigerator to prevent fermentation from occurring (so it doesnt become sour)

Nitrogen Fixation Nitrate reduction nitrite NO N2O N2 Nitrogen fixation N2 NH3

o Nitrogenase is the enzyme for this o Ammonia is then used for biosynthesis Oxidation and reduction o Oxidation produces energy o Reduction put energy in Nitrogen fixation is expensive, but if its the only way you can get it, then you have no choice, because you need nitrogen o Diazotrophs: bacteria and archaea that can fix nitrogen o Nitrogenase is O2 sensitive If you are strict anaerobe: youre good If you are a facultative anaerobe/strict aerobe, you may live around O2, which makes it hard to fix nitrogen Free-living cyanobacteria (Anabaena) o Blue-green algae o Filamentous o Photoautotrophs Get energy from sun, and get carbon from CO2 (fix CO2 and put into organic molecules, and fix N2 as well) Bacteria that live in symbiotic relationships with plant roots o When do photosynthesis, they make oxygen + glucose Takes water and CO2 energy, O2, and glucose (carbs) Have a particular cell type that differentiates into a cell called a heterocyst (does not do photosynthesis), specialized to do nitrogen fixation in a semianaerobic environment Grow them in minimal medium + nitrate o Dont need to work, so no heterocysts (or at least less) Grow them in minimal medium nitrate o Now they need to start fixing nitrogen, so you expect to see more heterocyst o The carbon source is CO2, none in the medium (since they can fix CO2) Helps cut down the number of organisms living in there o We need to give them light Can see with phase contrast and brightfield Akinete: functions similarly to a spore (metaboilicallly dormant), granular, largest cells