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

Biotechnology Explorer™ | explorer.bio-rad.com 2 Instructors - Bio-Rad Curriculum and Training Specialists Sherri Andrews, Ph.D. Damon Tighe, Leigh Brown, M.A.

Biotechnology Explorer™ | explorer.bio-rad.com 3 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!)

Biotechnology Explorer™ | explorer.bio-rad.com 4 A Case Study of doing a Case Study!  Study the impact of transformation temperature on transformation efficiency

Biotechnology Explorer™ | explorer.bio-rad.com 5 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 temperature It was on my lab bench…

Biotechnology Explorer™ | explorer.bio-rad.com 6 The Quick and Dirty continued… Heat shocked at 42ºC Heat shocked at 52ºC LB/Amp/Ara Plates 42ºC result52ºC result N=3 for each condition ?

Biotechnology Explorer™ | explorer.bio-rad.com 7 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?

Biotechnology Explorer™ | explorer.bio-rad.com 8 Results 42ºC Heat shock 60ºC Heat shock 75ºC Heat shock N=3 for each condition ???

Biotechnology Explorer™ | explorer.bio-rad.com 9 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

Biotechnology Explorer™ | explorer.bio-rad.com 10 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

Biotechnology Explorer™ | explorer.bio-rad.com 11 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

Biotechnology Explorer™ | explorer.bio-rad.com 12 STEM – Science  Understanding normal living conditions of HB101 E. coli  Understanding cell membrane structures  Understanding transformation and how it works

Biotechnology Explorer™ | explorer.bio-rad.com 13 STEM – Technology  Measurement devices  Heating implements  Calculation and analysis

Biotechnology Explorer™ | explorer.bio-rad.com 14 STEM – Engineering  Heat transfer  Conduction  Convection  Thermal conductivity

Biotechnology Explorer™ | explorer.bio-rad.com 15 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

Biotechnology Explorer™ | explorer.bio-rad.com 16 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

Biotechnology Explorer™ | explorer.bio-rad.com 17 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 MaterialThermal conductivity (W/m  K) at 298K=25ºC Air0.024 Aluminum205 Glass1.05 Polypropylene (plastic) Sand, dry Sand, moist Water0.58

Biotechnology Explorer™ | explorer.bio-rad.com 18 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…

Biotechnology Explorer™ | explorer.bio-rad.com 19 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 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

Biotechnology Explorer™ | explorer.bio-rad.com 20 STEM – Math Calculate rates of heat change using the different heating devices Heating deviceºC/second Heat block0.49 Water bath0.75 Sand bath0.42

Biotechnology Explorer™ | explorer.bio-rad.com 21 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 = µg Transformation efficiency 60 CFU µg = = 60,000 CFU/µg = 6 x 10 5 CFU/µg

Biotechnology Explorer™ | explorer.bio-rad.com 22 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 42ºC38.2ºC at 50 seconds100 42ºC40.9ºC at 1 min 40 seconds75 42ºC41.9ºC at 2 min 30 seconds63 60ºC51.7ºC at 50 seconds266 60ºC59.0ºC at 1 min 40 seconds3 60ºC61.0ºC at 2 min 30 seconds0

Biotechnology Explorer™ | explorer.bio-rad.com 23 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!

Biotechnology Explorer™ | explorer.bio-rad.com 24 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?