Transcript pGLO — STEM It Up! - Bio-Rad

STEMming up pGlo™
Bio-Rad Biotechnology Explorer™ pGlo Kit
Instructors - Bio-Rad Curriculum and Training
Specialists
Sherri Andrews, Ph.D.
[email protected]
Damon Tighe,
[email protected]
Leigh Brown, M.A.
[email protected]
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The Problem and the Constraints
 The Problem
– Develop a STEM lab experience using the pGlo kit
 The Contraints
– Personnel
– Time – Must be done by NSTA Nationals
– Money – Must not require lots of expensive equipment or
reagents
– Fun factor – Must be fun and interesting (at least to the
personnel doing it and hopefully also to educators and
students!)
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A Case Study of doing a Case Study!
 Study the impact of transformation temperature on
transformation efficiency
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The Quick and Dirty
 Time was one of the personnel’s biggest constraints so
designed quick and simple experiment to detect impact of
transformation temperature on transformation efficiency
Heat shock at 42ºC or 52ºC for 50
seconds, place on ice for 2 minutes
+
Digital dry bath
2 ml tube block
Why use the dry bath?
It’s digital and easy to set the
temperature
It was on my lab bench…
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42ºC Control
52ºC Test
The Quick and Dirty continued…
Heat
shocked
at 52ºC
Heat
shocked
at 42ºC
LB/Amp/Ara Plates
Average number of colonies
Impact of Heat Shock Temperature
350
300
250
200
150
100
50
0
42°C Heat shock
42ºC result 52ºC result
N=3 for each condition
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?
52°C Heat shock
When in doubt…repeat
 Theory – 52ºC was not quite warm enough to kill off
the E. coli so still got decent transformation
 Experiment – More is better! Increase heat shock
temperature to 60ºC or even 75ºC!
Heat shock at 42ºC or 60ºC or 75ºC for 50
seconds, place on ice for 2 minutes
+
Digital dry bath
2 ml tube block
What do you predict the results will be?
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Results
42ºC Heat shock
60ºC Heat shock
75ºC Heat shock
N=3 for each condition
Number of colonies
Im pact of Transform ation Tem perature
1600
1400
1200
1000
800
600
400
200
0
???
42°C Heat shock
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60°C Heat shock
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75°C Heat shock
What is going on???
 For HB101 E. coli, heat shock is most efficient at
42ºC (published results)
 HB101 E. coli should die at 75ºC!
 Using a dry bath, the best transformation efficiency
occurred when the temperature of the block was set
to 75ºC
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Experimental – What is impacting transformation
efficiency in my experiments?
 Design, construct and test a system which will
result in the best transformation efficiency using
what you know about thermal energy transfer
 Possible devices include
– Water bath, glass beaker, styrofoam cup, plastic cup
containing water at a specified temperature
– Dry bath
– Sand bath maintained at a certain temperature
– Incubator at a set temperature
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STEM in Action
 Problem – Increase the transformation efficiency of
the pGlo plasmid into HB101 by experimenting with
heat shock transformation conditions
 Constraints – Use equipment and reagents already in
existence in the laboratory or which can be
inexpensively built
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STEM – Science
 Understanding normal
living conditions of
HB101 E. coli
 Understanding cell
membrane structures
 Understanding
transformation and how
it works
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STEM – Technology
 Measurement devices
 Heating implements
Impact of Heat Shock Temperature
Average number of colonies
 Calculation and analysis
350
300
250
200
150
100
50
0
42°C Heat shock
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52°C Heat shock
STEM – Engineering
 Heat transfer
 Conduction
 Convection
 Thermal conductivity
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Test the device and calculate transformation
efficiency
 Test the device to see if transformation occurs and
determine efficiency of transformation
 If no transformation occurs, identify reasons which
impacted efficiency
 Identify and control variables such as number of E.
coli, amount of plasmid (loop vs. pipet), temperature
of heat shock, instruments used
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Develop Models
Measure the temperature of 250 µl Transformation
solution after 50 seconds when heated in the device
being used
50ºC
60ºC
All instruments were set to 75ºC
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45ºC
Develop models continued



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Measure the temperature a series of
locations of a frozen item thawing in
a warm location
Measure the temperature of a wet
item in a cold location and then in a
cold location with wind blowing over
it (or talk about wind chill factors)
Look up and discuss thermal
conductivity values
Measure the temperature of sand
warmed from above at different
heights from the surface
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Material
Thermal conductivity
(W/mK) at 298K=25ºC
Air
0.024
Aluminum
205
Glass
1.05
Polypropylene
(plastic)
0.1-0.22
Sand, dry
0.15-0.25
Sand, moist
0.25-2
Water
0.58
Develop models
 Crisco or coconut oil on top of water with a washer on
top
 Heat up
 Cool down
 See what happens!
 Insert photos here…
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STEM – Math
 Calculate conductive heat transfer through water versus sand if
the Transformation solution is at 20ºC, the water bath is 42ºC,
the wall thickness is 0.002 m, and the thermal conductivity is
assumed to be just based on the properties of sand or water
q / A = k dT / s
where
q / A = heat transfer per unit area (W/m2)
k = thermal conductivity (W/mK)
dT = temperature difference (ºC)
s = wall thickness (m)
 Calculate heat transfer per unit area through water if the water
temperature is set to 42ºC versus 60ºC
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STEM – Math
Calculate rates of heat change using the different
heating devices
Temperature change of 250 µl of Transformation
Solution Heated in 2 ml tubes
Heating device ºC/second
Temperature in °C
70
60
50
Heat block
40
30
0.49
Water bath
0.75
Sand bath
0.42
Water bath
Sand bath
20
10
0
0
20
40
60
Time in seconds
20
Heat block
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STEM – Math
 Calculate the transformation efficiency
– Count the number of colonies on the plate = CFU (colony forming units)
For this example, let’s assume you counted 60 colonies
– Determine the amount of DNA (in µg) spread on the plate
Volume spread (µl) x DNA in transformation (µg)
DNA spread on
=
the plate (µg)
Total volume of transformation (µl)
10 µl x 0.05 µg
500 µl
= 0.001 µg
– Finally, calculate the transformation efficiency by dividing the number of
colonies on the plate (CFU) by the amount of DNA (in µg) spread on the
plate
=
Transformation
60 CFU = 60,000 CFU/µg = 6 x 105 CFU/µg
=
0.001 µg
efficiency
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Final results of STEM experiment…
 Better controlled experiment
– Prepared one tube of resuspended cells in transformation solution
and aliquoted to several tubes to insure same cell density in each
– Using model system of just transformation solution, measured
temperature that cells would reach for heat shock
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Waterbath
setting
Measured temperature inside tube at
specific time
Number of transformed
colonies
42ºC
38.2ºC at 50 seconds
100
42ºC
40.9ºC at 1 min 40 seconds
75
42ºC
41.9ºC at 2 min 30 seconds
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60ºC
51.7ºC at 50 seconds
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60ºC
59.0ºC at 1 min 40 seconds
3
60ºC
61.0ºC at 2 min 30 seconds
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Some conclusions
 HB101 in this case had a higher transformation
efficiency in a waterbath set at 60ºC for 50 seconds
of heat shock
 HB101 had decreasing transformation efficiency if
allowed to sit at a higher temperature for too long
 The heating implement can have a direct impact on
the results due to heat exchange properties!
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Relation to other science and the real world
 Why do we have to study this??
–
–
–
–
PCR thin walled tubes
Wind chill factor (convection versus conduction)
Convection ovens (how come you can cook faster?)
Do you get colder if you are standing outside without a jacket
when it is -10ºC versus +10ºC? Why? What is the driving
force to become colder?
– Why does the USDA recommend cooking hamburger meat
until 160ºF (71ºC)? What temperature kills bacteria?
– What it the purpose of a fever? What temperature kills
bacteria? What is thermal death time versus thermal death
point?
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