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Amit Frishberg Lab: Dr. Iftach Nachman. Few facts about yeasts Can appear in haploid or diploid...
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Transcript of Amit Frishberg Lab: Dr. Iftach Nachman. Few facts about yeasts Can appear in haploid or diploid...
Amit Frishberg
Lab: Dr. Iftach Nachman
Few facts about yeasts
• Can appear in haploid or diploid forms.
• Can grow on different types of carbon
mediums.
• Can use respiration or fermentation as
their preferred carbon consuming method.
• Have a relatively high reproduction rate.
Respiration / Fermentation
Respiration
Carbon
Glycolysis
Aerobic conditions
Fermentation
Carbon
Glycolysis
Anaerobic conditions
Ethanol
The selected method depends on the carbon medium which the yeasts use.
Can be reused by the yeasts
Experiments in the lab
• Researching the differences in the nutrient
sensing mechanism in different types of
wild type yeasts.
• Finding the genotypic differences in those
types that cause to the diverse in the
mechanism.
My project questions
• Is there a difference in the growth rate of
different types of yeasts (S. cerevisiae)?
• What are the reasons for those differences if
they’re exist?
• How do different carbon mediums effect the
growth rate of different types of yeasts?
So where did we start?
55gift from Barak CohenYPS606Oak Tree
56gift from Barak CohenYPS623Oak Tree
57gift from Barak CohenBW-1Oak Tree
58gift from Barak CohenCP-1Oak Tree
59gift from Barak CohenIN-1Oak Tree
60gift from Barak CohenT7Oak Tree
61gift from Barak CohenTN-1Oak Tree
62gift from Barak CohenNC-02Oak Tree
63gift from Barak CohenIL-01Oak Tree
64gift from Kruglyak's labCLIB192 Baker France
65gift from Kruglyak's labCLIB274_1
66gift from Kruglyak's labCLIB326_1
67gift from Kruglyak's labCLIB219_2b
68gift from Kruglyak's labCLIB272_2
69gift from Kruglyak's labCLIB382 Beer Ireland
70gift from Kruglyak's labYJM145 Clinical. AIDS patient Unites States
71gift from Kruglyak's labYJM280 Clinical. peritoneal fluid Unites States
72gift from Kruglyak's labYJM320b Clinical. blood Unites States
73gift from Kruglyak's labYJM326 Clinical. unknown Unites States
74gift from Kruglyak's labYJM413 Clinical. blood Unites States
75gift from Kruglyak's labYJM421 Clinical. ascites fluid Unites States
76gift from Kruglyak's labYJM428 Clinical. paracenteis fluid Unites States
34gift from Barak Cohen
UCD 51Vineyard
35
gift from Barak Cohen
UCD 175Vineyard
36
gift from Barak Cohen
UCD 522Vineyard
37
gift from Barak Cohen
UCD 529Vineyard
38
gift from Barak Cohen
UCD 762Vineyard
39
gift from Barak Cohen
UCD 765Vineyard
40
gift from Barak Cohen
UCD 781Vineyard
41
gift from Barak Cohen
UCD 820Vineyard
42
gift from Barak Cohen
UCD 2120Vineyard
43gift from Barak CohenM5Vineyard
44gift from Barak CohenM7Vineyard
45gift from Barak CohenM13Vineyard
46gift from Barak CohenM15Vineyard
47gift from Barak CohenM22Vineyard
48gift from Barak CohenM29Vineyard
49gift from Barak CohenM30Vineyard
50gift from Barak CohenM33Vineyard
51gift from Barak CohenM34Vineyard
52YIN1-Msn2-482yIN1 transformed with PCR product of msn2 and pSR482
53YIN1-Msn4-482yIN1 transformed with PCR product of msn4 and pSR482
54YIN1-Rim15-482
yIN1 transformed with PCR product of rim15 and pSR482
And many more…
We had more than 100 species to begin with:
Problems with the system
• The experiment was too complicated (too many
species) - it would have take us too much time.
• We wanted to focus on the most important
species in order to study them better.
• We didn’t know which medium to use for most of
our species.
Narrowing the species
• We selected 20 types of yeasts:
– Wine yeasts - 9 types (34-42).
– Oak yeasts – 9 types (55-63).
– Sk1 , sigma (lab strains)
• We wanted to compare between the well
researched wine yeasts and the oak yeasts.
Checking OD for growth rate
1. Setting the basic OD of all species to
the same level.
2. Monitoring the change in OD over time.
3. Managing the result (using excel and
mathlab) to build growth charts.
Work method
20 species in test tubes – non saturated
Raising over night (O.N)
20 species in test tubes – saturated
Dilution to a 96 cells plate
Putting inside the robot
Structure of the 96 cells plate
123456789101112
B 57 616335SK139 A
B555759616335SK139 B
B555759616335SK139 C
B554259414141sigmasigma D
B56424262373737sigma E
B5658606234363840 F
B5658606234363840 G
B 5860 34363840 H
Using the robot
• The robot contains 3 main parts:
1. The incubator.
2. The shaker.
3. The OD reader.
30 min incubation
1 min shake
OD check
תמונה של
הרובוט
Growth rate on Glucose rich medium (YPD)
• We assumed that both types of
yeasts will use this medium and grow
fast on it.
• We put the 96 cells plate for 24 hours
in the robot.
ResultsOD as a function of time
ResultslogOD as a function
of time
ResultsOD mean as a function
of time
ResultslogOD mean as a function
of time
Conclusions
• Both types of yeasts got to the same saturation levels.
• Both types of yeasts used fermentation as their
preferred carbon consuming method.
• Oak yeasts got out of lag phase faster than wine
yeasts.
• Oak yeasts had a faster growth rate than wine yeasts.
Growth rate on Acetate rich medium (YPA)
• We assumed that both types of yeasts will use
this medium and grow on it.
• We assumed that oak yeasts will grow faster on
it than wine yeasts.
• We put the 96 cells plate for 72 hours in the
robot.
ResultsOD as a function of time
ResultslogOD as a function
of time
ResultsOD mean as a function
of time
ResultslogOD mean as a function
of time
Conclusions
• The growth rate of both types of yeasts was much slower
than on YPD.
• Both types of yeasts used fermentation as their preferred
carbon consuming method.
• Both types of yeasts got almost to the same saturation
levels.
• Oak yeasts got out of lag phase faster than wine yeasts.
• Oak yeasts had a faster growth rate than wine yeasts.
Does cell size take part in those results?
Both types of yeasts has almost the same cell size
We observed the two types of yeasts with an electron microscope:
Oak yeastsWine yeasts
The clumpiness factor
• Both type of yeasts were capable of creating
clumps.
• The wine yeasts had a much higher clumpiness
ratio than the oak yeasts.
Oak yeasts
Wine yeasts
Future experiment
• Studying the biological mechanism that
provides the oak yeasts a faster growth
rate on those mediums.
• Continue the comparison between wine
and oak yeasts to different types of yeasts.
Thanks
Dr. Iftach Nachman
Dr. Yifat Goldshmidt