AP BIOLOGY Investigation #8 Biotechnology: Bacterial Transformation - PDF document

Slide 1 / 31 New Jersey Center for Teaching and Learning Progressive Science Initiative This material is made freely available at www.njctl.org and is intended for the non-commercial use of students and teachers. These materials may not be used for any commercial purpose without the written permission of the owners. NJCTL maintains its website for the convenience of teachers who wish to make their work available to other teachers, participate in a virtual professional learning community, and/or provide access to course materials to parents, students and others. Click to go to website: www.njctl.org Slide 2 / 31 AP BIOLOGY Investigation #8 Biotechnology: Bacterial Transformation Summer 2014 www.njctl.org Slide 3 / 31 Investigation #8: Transformation Click on the topic to go to that section · Pacing/Teacher's Notes · Pre-Lab · Guided Investigation · Independent Inquiry

Slide 4 / 31 Pacing/Teacher's Notes Return to Table of Contents Slide 5 / 31 Teacher's Notes Lab procedure adapted from College Board AP Biology Investigative Labs: An Inquiry Approach Teacher's Manual Click here for CB AP Biology Teacher Manual Slide 6 / 31 Pacing General Reference to Day (time) Activity Notes Description Unit Plan Day 1 (HW) GE Day 9 Pre-lab Background Prepare plates for tomorrow HW Be sure to review instructions for Guided Transformation your plasmid, and add any Day 2 (80) GE Day 10 Practice & Plating necessary steps. Incubate plates overnight Calculating Day 3 (40) Analysis Transformation GE Day 11 Efficiency Independent Transformation Day 4 (40) GE Day 12 Inquiry & Plating Calculating Independent Transformation Day 5 (40) GE Day 13 Efficiency & Inquiry Discussion Day 6 (20) Assessment Lab Quiz GE Day 14

Slide 7 / 31 Pre-Lab Return to Table of Contents Slide 8 / 31 Question/Objectives How can we use genetic engineering techniques to manipulate heritable information? In this lab we will: · Demonstrate the universality of DNA and its expression. · Explore the concept of phenotype expression in organisms. · Explore how genetic information can be transferred from one organism to another. · Investigate how horizontal gene transfer is a mechanism by which genetic variation is increased in organisms. · Explore the relationship between environmental factors and gene expression. · Investigate the connection between the regulation of gene expression and observed differences between individuals in a population of organisms. Slide 9 / 31 Pre-Lab Questions Read the background information and answer the following questions in your lab notebook. 1. What causes mutations in bacteria? Can mutations affect plasmids? 2. What is the function of plasmids in bacteria? 3. Do cells take up more plasmids in some conditions and less in others?

Slide 10 / 31 Pre-Lab Questions Read the "Getting Started" and answer the following questions in your lab notebook. 1. Some bacteria are naturally resistant to antibiotics, but others are not. How could you use two LB/agar plates, so E. coli , and some ampicillin (an antibiotic) to determine how E. coli cells are affected by ampicillin? 2. What would you expect your experimental result to indicate about the effect of ampicillin on the E. coli cells? Do you think that exposure to ampicillin will cause the E. coli cells to evolve resistance to ampicillin? Why or why not? 3. How will you be able to tell if host E. coli cells have been genetically transformed? Slide 11 / 31 Safety When working with a culturing bacteria, it is important not to introduce contaminating bacteria or fungi into the experiment. Because these microorganisms are ubiquitous, i.e., you can find them everywhere - on fingertips, bench tops, lab tables, etc. - you must avoid these contaminating surfaces. When working with inoculation loops, bulb pipettes, micropipettes, and agar plates, do not touch the tips of them (or in the case of agar, the surface itself) or place them directly onto contaminating surfaces. Be sure to wash your hands before beginning the procedure and after - and cover your sneezes. Do not eat, drink, apply cosmetics, or use personal electronic devices in the work area. Slide 12 / 31 Guided Investigation Return to Table of Contents

Slide 13 / 31 Materials Your Workstation · Transformation solution (CaCl 2 ) · E. coli starter plate · Microcentrifuge tubes and holder · 2 LB agar plates · Container of crushed ice · 2 LB/amp agar plates · Marking pen · LB nutrient broth · Lab notebook · Sterile inoculation loops · 100-1000 microliter bulb pipettes · 1-10 microliter micropipettes with sterile tips Common Workstation · DNA plasmid · 42 o C water bath and thermometer · 37 o C incubator · 20 microliter adjustable-volume micropipette · 10% household bleach · Biohazardous waste disposal bags · Masking or lab tape Slide 14 / 31 Guided Practice Step 1 Familiarize yourself with sterile technique before beginning the experiment. Step 2 Label one closed microcentrifuge tube "+plasmid" and the other tube "-plasmid". Label both tubes with your group's number, and place them in the microcentrifuge tube holder. Step 3 Carefully open the tubes and using a 100-1000 microliter bulb pipette with a sterile tip, transfer 250 microliter of the ice cold transformation solution into each tube. Step 4 Place both tubes on (into) the ice. Slide 15 / 31 Guided Practice Step 5 Use a sterile inoculation loop to pick up a single colony of bacteria from your starter plate. Be carful not to scrape off any agar from the plate. Pick up the "+plasmid" tube and immerse the loop in the CaCl 2 solution at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the solution. Appropriately discard the loop. Step 6 Use a new sterile 100-1000 micropipette to repeatedly pulse the cells in solution to thoroughly resuspend the cells. Place the tube back on the ice. Step 7 Using a new sterile inoculation loop, repeat Steps 5 and 6 for the "-plasmid" tube.

Slide 16 / 31 Guided Practice Step 8 Using a 1-10 microliter micropipette with a sterile tip, transfer 10 microliters of plasmid solution directly into the E. coli suspension in the "+plasmid" tube. Tap tube with a finger to mix, but avoid making bubbles in the suspension or splashing the suspension up the sides of the tube. Do not add the plasmid into the "-plasmid" tube! Step 9 Incubate both tubes (+plasmid and -plasmid) on ice for 10 minutes. Make sure the bottom of the tubes make contact with the ice. Step 10 While the tubes are sitting on ice, label each of your agar plates on the bottom (not the lid). Slide 17 / 31 Guided Practice Step 11 Following the 10-minute incubation at 0 o C, remove the tubes from the ice and "heat shock" the cell in the tubes. It is critical that the cells receive a sharp and distinct shock! Make sure the tubes are closed tightly! Place the tubes into a test tube holders float, and dunk the tubes into the water bath, set at 42 o C, for exactly 50 seconds. Make sure to push the tubes all the way down in the holder so that the bottom of the tubes with the suspension makes contact with the warm water. Step 12 When the 50 seconds have passed, place both tubes back on ice. For best transformation results, the change from 0 o C to 42 o C and then back to 0 o C must be rapid. Incubate the tubes on ice for an additional two minutes. Slide 18 / 31 Guided Practice Step 13 Remove the holder containing the tubes from the ice and place on lab counter. Using a 100-1000 microliter micropipette with sterile tip, transfer 250 microliters of LB nutrient broth to the "+plasmid" tube. Close the tube and gently tap with your finger to mix. Repeat with a new sterile micropipette for the "-plasmid" tube. Step 14 Incubate each tube for 10 minutes at room temperature. Step 15 Use a 10-1000 microliter micropipette with sterile tip to transfer 100 microliters of the transformation (+plasmid) and the control (-plasmid) suspensions onto the appropriate LB and LB/ amp plates. Be sure to use a separate pipette for each of the four transfers.