Growth Curve Experiment
Chemical transformation of yeast requires the organism to be in logarithmic growth phase, so, before we clone our casein genes into yeast, we need to make sure that we know how to grow our yeast to log Phase. Also, this data will elucidate the effects of heterologous protein expression on our strains.
Here's a brief protocol for monitoring yeast growth.
(1) Figure out growth curve of yeast strains (we have Carolina strain now, we want ATCC strain, and Keoni Strain) (a) Streak Carolina strain on a YPD + Ura plate for isolated colonies. (b) Grow overnight at 30C (c) Pick a single colony from the plate (in triplicate) and grow in 5mL YPD + Ura (d) The next day, Record the OD (650nm) of the >16hour culture, and dilute the Growth Curve Culture by adding 100uL of 16hour culture to 99.9mL of YPD + Ura. (e) Grow at 30C, 250rpm. (e) Record the OD (650nm) at 0, 5, 10, 24, 48, and 72 hours. Use YPD + Ura as the blank for each reading.
(2) Repeat growth curve with pD1214:FAKS-hKappa cloned into yeast strain. (a) no, it's not the real human kappa casein, but it'll get us closer to understanding how our strains will perform after real cloning happens. This is a great set of experiments for folks who've not done cloning before. Please learn about Bacterial transformation and plating for single colonies, Plasmid Prep, and Yeast transformation. (b) Transform positive plasmid into E. coli (see Molecular Biology page) (c) Instead of plating transformation, add the transformation reaction to 5mL of LB + Carb(50). (d) Grow this plasmid overnight at 37C, rotating; and plasmid prep the DNA the next day. (e) Transform yeast using the Yeast Transformation protocol. (f) Plate transformation and incubate overnight at 30C. (g) Select single colonies and repeat from (1) - (c) (above).
(3) This experiment can be repeated with different starting concentrations of Yeast culture (1:2, 1:10, 1:50, 1:100, 1:500, 1:1000).
(4) RECORD YOUR DATA, MAKE A FIGURE, and POST IT HERE.
Failed Growth Curve Experiments: Advait, Johan, Minakshi, Lafia, Shashank
Day 1, 8/22/14 1) Streaked URA3 deficient yeast (from Shashank) onto plates. 2) Measured 0.603 grams active dry yeast into 59 ml pure distilled water 3) Microwaved water with yeast for 10-12 seconds and then put yeast at 37 degrees celsius to activate 4) Streak plates with active dry yeast 5) Incubate plates at 30 degrees celsius, and measure growth after two hours 6) Now there are 2 active dry yeast plates and 2 URA3 deficient yeast plates
Day 2, 8/23/14 1) Took 6 single colonies from the plates and placed each in tubes with nutrient broth (there are 3 colonies from active dry yeast and 3 from URA3 yeast 2) Placed the 6 tubes in the Flex Chem oven. 3) Autoclaved containers
Day 3, 9/24/14 1) Took OD Spec readings: A) (Blank) 0.0000 nm -> 0% B) (sample 1 yeast URA3) 0.2416 nm -> 24.16% C) (sample 2 yeast URA3) 0.2570 nm -> 25.70%
Future Spec Readings: Aaron took hemocytometer readings later, and he got: 4.6 x 10^6 cells/ml of culture. (Need to confirm this reading with Aaron) Optimal Log phase is 2.7 x 10^7 cells/ml Add spectrophotometer readings here: https://docs.google.com/spreadsheets/d/16RZMZfr_rv6GlAPxfWJlHP5ca3nTfZUWq57frVQsKiI/edit#gid=0