STEMming up pGlo Bio-Rad Biotechnology Explorer pGlo Kit
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] 2 Biotechnology Explorer | explorer.bio-rad.com 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!) 3 Biotechnology Explorer | explorer.bio-rad.com A Case Study of doing a Case Study! Study the impact of transformation temperature on transformation efficiency 4 Biotechnology Explorer | explorer.bio-rad.com The Quick and Dirty Time was one of the personnels biggest constraints so designed quick and simple experiment to detect impact of transformation temperature on transformation efficiency
Heat shock at 42C or 52C for 50 seconds, place on ice for 2 minutes + Digital dry bath 2 ml tube block Why use the dry bath? Its digital and easy to set the temperature It was on my lab bench 5 Biotechnology Explorer | explorer.bio-rad.com 42C Control 52C Test The Quick and Dirty continued Heat shocked
at 52C Heat shocked at 42C LB/Amp/Ara Plates Average number of colonies Impact of Heat Shock Temperature 350 300 250 200 150 100 50 0 42C Heat shock 42C result 52C result N=3 for each condition 6
Biotechnology Explorer | explorer.bio-rad.com ? 52C Heat shock When in doubtrepeat Theory 52C 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 60C or even 75C! Heat shock at 42C or 60C or 75C for 50 seconds, place on ice for 2 minutes + Digital dry bath 2 ml tube block What do you predict the results will be? 7
Biotechnology Explorer | explorer.bio-rad.com Results 42C Heat shock 60C Heat shock 75C 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 42C Heat shock
8 60C Heat shock Biotechnology Explorer | explorer.bio-rad.com 75C Heat shock ??? What is going on??? For HB101 E. coli, heat shock is most efficient at 42C (published results) HB101 E. coli should die at 75C! Using a dry bath, the best transformation efficiency occurred when the temperature of the block was set to 75C 9 Biotechnology Explorer | explorer.bio-rad.com
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 10 Biotechnology Explorer | explorer.bio-rad.com 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
11 Biotechnology Explorer | explorer.bio-rad.com STEM Science Understanding normal living conditions of HB101 E. coli Understanding cell membrane structures Understanding transformation and how it works 12 Biotechnology Explorer | explorer.bio-rad.com 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 42C Heat shock 13 Biotechnology Explorer | explorer.bio-rad.com 52C Heat shock
STEM Engineering Heat transfer Conduction Convection Thermal conductivity 14 Biotechnology Explorer | explorer.bio-rad.com 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 15 Biotechnology Explorer | explorer.bio-rad.com
Develop Models Measure the temperature of 250 l Transformation solution after 50 seconds when heated in the device being used 50C 60C All instruments were set to 75C 16 Biotechnology Explorer | explorer.bio-rad.com 45C Develop models continued
17 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 Biotechnology Explorer | explorer.bio-rad.com Material
Thermal conductivity (W/mK) at 298K=25C 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 18 Biotechnology Explorer | explorer.bio-rad.com STEM Math Calculate conductive heat transfer through water versus sand if
the Transformation solution is at 20C, the water bath is 42C, 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 (C) s = wall thickness (m) Calculate heat transfer per unit area through water if the water temperature is set to 42C versus 60C 19 Biotechnology Explorer | explorer.bio-rad.com STEM Math Calculate rates of heat change using the different heating devices Temperature change of 250 l of Transformation Solution Heated in 2 ml tubes
Heating device C/second Temperature in C 70 60 50 Heat block 40 30 Water bath Sand bath 20 10 0 0 20
40 60 Time in seconds 20 Biotechnology Explorer | explorer.bio-rad.com 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, lets assume you counted 60 colonies Determine the amount of DNA (in g) spread on the plate Volume spread (l) x DNA in transformation (g) DNA spread on = the plate (g) Total volume of transformation (l) = = 10 l x 0.05 g 500 l 0.001 g Finally, calculate the transformation efficiency by dividing the number of colonies on the plate (CFU) by the amount of DNA (in g) spread on the plate Transformation 60 CFU = 60,000 CFU/g = 6 x 105 CFU/g = 0.001 g
efficiency 21 Biotechnology Explorer | explorer.bio-rad.com 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 22 Waterbath setting Measured temperature inside tube at specific time Number of transformed colonies
42C 38.2C at 50 seconds 100 42C 40.9C at 1 min 40 seconds 75 42C 41.9C at 2 min 30 seconds 63 60C 51.7C at 50 seconds
266 60C 59.0C at 1 min 40 seconds 3 60C 61.0C at 2 min 30 seconds 0 Biotechnology Explorer | explorer.bio-rad.com Some conclusions HB101 in this case had a higher transformation efficiency in a waterbath set at 60C 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! 23 Biotechnology Explorer | explorer.bio-rad.com 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 -10C versus +10C? Why? What is the driving force to become colder? Why does the USDA recommend cooking hamburger meat until 160F (71C)? What temperature kills bacteria? What it the purpose of a fever? What temperature kills bacteria? What is thermal death time versus thermal death
point? 24 Biotechnology Explorer | explorer.bio-rad.com
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