Post on 23-Dec-2015
Biotechnology Explorer™ | explorer.bio-rad.com2
Instructors - Bio-Rad Curriculum and Training Specialists
Sherri Andrews, Ph.D.
sherri_andrews@bio-rad.com
Damon Tighe,
damon_tighe@bio-rad.com
Leigh Brown, M.A.
leigh_brown@bio-rad.com
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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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42ºC Control 52ºC Test
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
+
Digital dry bath 2 ml tube block
Heat shock at 42ºC or 52ºC for 50 seconds, place on ice for 2 minutes
Why use the dry bath?It’s digital and easy to set the temperatureIt was on my lab bench…
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The Quick and Dirty continued…
Heat shocked at 42ºC
Heat shocked at 52ºC
LB/Amp/Ara Plates
42ºC result 52ºC result
Impact of Heat Shock Temperature
0
50
100
150
200
250
300
350
42°C Heat shock 52°C Heat shock
Ave
rag
e n
um
ber
of
colo
nie
sN=3 for each condition
?
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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!
+
Digital dry bath 2 ml tube block
Heat shock at 42ºC or 60ºC or 75ºC for 50 seconds, place on ice for 2 minutes
What do you predict the results will be?
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Results
42ºC Heat shock 60ºC Heat shock 75ºC Heat shock
Impact of Transformation Temperature
0200400600800
1000120014001600
42°C Heat shock 60°C Heat shock 75°C Heat shock
Nu
mb
er o
f co
lon
ies
N=3 for each condition
???
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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
Calculation and analysisImpact of Heat Shock Temperature
0
50
100
150
200
250
300
350
42°C Heat shock 52°C Heat shock
Ave
rag
e n
um
ber
of
colo
nie
s
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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 45ºC
All instruments were set to 75ºC
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Develop models continued
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
Material Thermal conductivity (W/mK) 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
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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 / swhere 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
0
10
20
30
40
50
60
70
0 20 40 60
Time in seconds
Tem
per
atu
re i
n °
C
Heat block
Water bath
Sand bath
Heating device ºC/second
Heat block 0.49
Water bath 0.75
Sand bath 0.42
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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
– 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
DNA spread on the plate (µg)
Volume spread (µl) x DNA in transformation (µg)
Total volume of transformation (µl)=
= 10 µl x 0.05 µg
500 µl
= 0.001 µg
Transformation efficiency
60 CFU0.001 µg= = 60,000 CFU/µg = 6 x 105 CFU/µg
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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
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 63
60ºC 51.7ºC at 50 seconds 266
60ºC 59.0ºC at 1 min 40 seconds 3
60ºC 61.0ºC at 2 min 30 seconds 0
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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?