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TRANSFORMATION: Tips & Tricks For AP Biology Exploration 8Danielle R. Snowflack, Ph.D.www.edvotek.com
Slide2EDVOTEKThe Biotechnology Education CompanyCelebrating OVER 30 years
of science education!
Slide3www.edvotek.com/contestEnds January 31, 2021
Slide4Bacterial Transformation: AP08
Slide5Today’s Experiment: Bacterial TransformationIn the lab, we can program bacteria to take up genes that make them express important proteins, antibiotics, or biopharmaceuticals.Transformation of Bacteria with Green Fluorescent Protein (AP08/223)Clockwise from top right:Kits 222, 221, 224, and 301
Slide6Genes can be Transferred Between BacteriaIn conjugation, cells connect to transfer DNA from one to the other. In transformation, bacteria pick up DNA from the surrounding area. The newly acquired genetic information is both stable and heritable.
Slide7Plasmids pass genes between bacteriaSmall circular DNA molecules called plasmids allow bacteria to exchange beneficial genes. antibiotic resistancevirulence factorsnitrogen fixation
Slide8Recombinant plasmids are used for transformationOrigin of Replication: a DNA sequence that shows bacteria where to start copying a plasmid. Promoter: a DNA sequence that shows RNA polymerase where to start transcribing a gene.Multiple Cloning Site: a segment of DNA that contains sites where genes can be inserted.Selectable marker: a gene that allows researchers to identify transformed bacteria.
Slide9Transformation of bacteria by heat shockAfter DNA is added to the cells, the suspension is “heat shocked” — or moved quickly between widely different temperatures. The combination of chemical ions and the rapid change in temperature alters the permeability of the cell wall and membrane, allowing DNA molecules to enter the cell.
Slide10The NEW and IMPROVED transformation protocolPreparation of the Agar PlatesPreparation of the competent cells Resuspend the Bacteria in Cold CaCl2Add the Correct Amount of PlasmidTime and Temperature of Heat ShockLength of Recovery Period
Slide11Tip #1: Proper preparation of the LB-Agar platesThese petri plates contain a solid nutrient agar that we use to culture microorganisms.Reagents like IPTG and Ampicillin are heat sensitive, so be sure to cool the media to 60°C before adding.Prepare plates 2-7 days before performing the experiment.
Slide12Tip #2: Preparation of competent cellsCentrifuge E. coli cell culture samples for five minutes to pellet cells.Carefully pour off the supernatant without disturbing the cell pellet.Resuspend the cells in ice-cold CaCl2 by gently pipetting up and down several times. Incubate the tubes on ice for 10 minutes, then pellet the cells by centrifuging.Pour off supernatant, then gently resuspend cells in ice-cold Competent Cell Solution.
Slide13Tip #3: Keep everything cold!Pre-chill the reagents before starting the lab.Resuspend the cells by gently pipetting up and down until no clumps are visible. Cells must be completely resuspended or the plasmid will not contact the bacterial cell walls or membranes. Keep the cells cold before the heat shock. Hold the top of the tube to prevent heat transfer.
Slide14Tip #4: Add the correct amount of plasmid DNAAdding too much or too little plasmid DNA will reduce the transformation efficiency.Pipetting error can affect your student’s results. Make sure your students know how to accurately pipet before the experiment by practicing#S-44Edvotek MyLab™ 1106
Edvotek
Kit S-44
Slide15Tip #5: Time and temperature of heat shockFor best results, keep the cell suspension ice-cold before and after the heat shock.Use a thermometer to confirm that the water bath reaches 42C.Make sure the cells are only heat shocked for 45 seconds.
Slide16Tip #6: Let cells recover for 10 minutesDuring this incubation, the bacteria repair their cell walls and express the antibiotic resistance gene. If the cells are plated too soon, they may not be able to grow on the selective media. After 20-30 minutes of recovery, the bacteria will double, meaning that many of the "transformants" are produced by cell division.
Slide17Transformation of bacteria by heat shockADD DNA to tube and INCUBATE on ice for 10 min.PLACE the transformation tubes in a 42°C water bath for 45 sec. *The heat shock facilitates the entry of DNA into the E. coli.Immediately RETURN the tubes to the ice and INCUBATE for 2 min.TRANSFER 250L of Luria Recovery Broth to the tubes using a sterile one mL pipet. Gently MIX by flicking the tube.INCUBATE the cells for ten min in a 37° C water bath. *This recovery period allows cells to repair their cell walls and to express the antibiotic resistance gene.
SPREAD
the cells on selective agar media plates.
Slide18Control of gene expression with an inducible promoterInducible promoters allow precise control because expression of the gene will only “turn on” in the presence of a small molecule like arabinose, tetracycline, or IPTG.
Slide19Results of transformation experiment
Slide20Transformation efficiencyTransformation is highly inefficient – only one in every 10,000 cells successfully incorporates the plasmid DNASince each colony comes from a single transformed cell, we can determine the number of cells transformed per microgram of DNAQuick reference:50 ng (0.05µg) plasmid DNA0.5 mL recovery volume0.25 mL plated
Slide21Extension Activity: Independent InvestigationsHow can we change conditions to improve transformation efficiency? Amount of DNATime/temperature of heat shockDifferent salt (RbCl2 vs. CaCl2)Competent cells vs picking coloniesWhat effects do mutations have on gene expression?What happens if we transform multiple plasmids?Does the plasmid give a selective advantage over untransformed bacteria?
Slide22Extension Activity: Colony PCR (Edvotek Kit 323)PCR can confirm the presence of the plasmid DNA in the bacterial host.Any cells containing the GFP plasmid should give positive results, proving that the newly acquired genetic information is present in the cells.Using PCR, test cells from different plates, satellite colonies, even some pre-transformed bacteria.
Image from protocols.io
Slide23Extension Activity: Adding art to your experimentsIncorporating art or design into your transformation experiment can be as simple or involved as you would like to make it.Bacteria can become “living paint” for student art projects.Each year, ASM hosts an “Agar Art” competition. See examples of the winners here!
Kit 226
Slide24Extension activity: Bioethics discussionApplication of genetic engineering in the real worldEthical, social, and medical issues related to manipulation of DNA technologyNew DNA technologies and how they revolutionize genetic engineeringImage from: https://
blog.helix.com
/bioethics/
Slide25Today’s Experiment: Bacterial TransformationIn the lab, transformation is used to program bacteria to take up genes that make them build biofuels, biopharmaceuticals, fine chemicals, and even change color!The NEW competent cell prep will make your results more consistent.Bacterial transformation can be used as a starting activity to bring advanced biotechnology, bioethics, bioart, and more into your classroom.
Slide26www.edvotek.com/contestEnds January 31, 2021
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