stemming up pglo™ bio-rad biotechnology explorer™ pglo kit

STEMming up pGlo™Bio-Rad Biotechnology Explorer™ pGlo Kit

Biotechnology Explorer™ | explorer.bio-rad.com2

Instructors - Bio-Rad Curriculum and Training Specialists

Sherri Andrews, Ph.D.

[email protected]

Damon Tighe,

[email protected]

Leigh Brown, M.A.

[email protected]


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!)


A Case Study of doing a Case Study!

Study the impact of transformation temperature on transformation efficiency


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…


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

?


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?


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

???


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


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


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


STEM – Science

Understanding normal living conditions of HB101 E. coli

Understanding cell membrane structures

Understanding transformation and how it works


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


STEM – Engineering

Heat transfer

Conduction

Convection

Thermal conductivity


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


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


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


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…


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


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


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


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


60ºC 51.7ºC at 50 seconds 266




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!


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?

stemming up pglo™ bio-rad biotechnology explorer™ pglo kit

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