Difference between revisions of "Growth Curve Experiment"

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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.  
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.
Here's a brief protocol for monitoring yeast growth.


*'''GROWTH CURVE'''
'''GROWTH CURVE'''
(1) Figure out growth curve of yeast strains (we have Carolina strain now, we want ATCC strain, and Keoni Strain)  
(1) ''Figure out growth curve of yeast strains (we have Carolina strain now, we want ATCC strain, and Keoni Strain)''
     (a) please be conservative with YPD media here - we can do these experiments in 100mL         
     (a) Streak Carolina strain on a YPD + Ura plate for isolated colonies.
          tubes - in triplicate. Record the OD (650nm) at 0, 5, 10, 24, 48, and 72 hours.  
    (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) grow the yeast clones in triplicate (100mL) to create growth curve.


(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''').


Select a single colony and grow it overnight in 5mL YPD at 30C, rotating at 250rpm. Read the OD the next morning (>16hrs later). Record the OD at 16hours, then start the culture with a KNOWN amount of 16-hr innoculum.  
(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).


      (a) Dilute the 16hour overnight in YPD, or 0.1mL into 99.9mL (1:1000) YPD.
(4) ''RECORD YOUR DATA, MAKE A FIGURE, and POST IT HERE.''


       (b) Record the OD (650nm) at 0, 5, 10, 24, 48, and 72 hours.
 
 
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

Latest revision as of 03:57, 2 September 2014

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.

GROWTH CURVE

(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