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TEACHER’S MANUAL AND STUDENT GUIDE

Transformation

Carolina Investigations® for AP* Biology

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TEACHER’S MANUAL

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3Content Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3Time Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6Teaching Inquiry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7Background Information . . . . . . . . . . . . . . . . . . . . . . . . .8Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13Answers to Questions in the Student Guide . . . . . . . . .14Helpful Hints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20Extension Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20Related Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21

STUDENT GUIDE*

Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .S-1Pre-laboratory Inquiry . . . . . . . . . . . . . . . . . . . . . . . . . .S-5Transformation Laboratory Procedure . . . . . . . . . . . . .S-6

Laboratory Questions . . . . . . . . . . . . . . . . . . . . . . . .S-9Big Idea Assessments . . . . . . . . . . . . . . . . . . . . . . .S-12

AppendixSterile Technique . . . . . . . . . . . . . . . . . . . . . . . . . .S-14Data Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .S-15

*Photocopy the Student Guide as needed for use in your classroom.

The materials and activities in this kit meet the guidelines and academicstandards of the Advanced Placement (AP) Program and have beenprepared by Carolina Biological Supply Company, which bears soleresponsibility for kit contents.

Table of Contents

©2014 Carolina Biological Supply Company/Printed in USA.

©Carolina Biological Supply Company/Printed in USA. 3

Teacher’s Manual

NOTESCarolinaTM Transformation Kit for AP BiologyOverviewThe goal of this lab is to familiarize students with the transformation of bacteria byplasmids, a key technique in genetic engineering. The process of transformation isalso a powerful demonstration of the role of DNA in determining phenotype.Students will be asked to design an experiment to distinguish between three distinct plasmids by determining which of three genes are present on whichplasmids. Instructor-led discussions will ensure that these experiments are designed appropriately. The 1-station kit is designed for one group. The 8-station kit is designed for 8 groups of students, with the number of students in each group being determined by the instructor.

ObjectivesStudents will

• become familiar with experiments leading to the identification of DNA as the molecule that passes on traits.

• transform bacteria using plasmid DNA.

• perform an experiment demonstrating the role of DNA in determiningphenotype.

• learn what a plasmid is.

• learn about one mechanism of antibiotic resistance.

• be introduced to a powerful model of natural selection.

• learn about horizontal gene transfer.

Content StandardsThis kit is appropriate for Advanced Placement high school students andaddresses the following AP Biology concepts:

Big Idea #1: The process of evolution drives the diversity andunity of life.

• Essential Knowledge 1.A.1: Natural selection is a major mechanism ofevolution.

• Essential Knowledge 1.A.2: Natural selection acts on phenotypic variationsin populations.

Big Idea #3: Living systems store, retrieve, transmit, andrespond to information essential to life processes.

• Essential Knowledge 3.A.1: DNA and in some cases RNA, is the primarysource of heritable information

• Essential Knowledge 3.C.1: Changes in genotype can result in changes inphenotype.

• Essential Knowledge 3.C.2: Biological systems have multiple processes thatincrease genetic variation.

4 ©Carolina Biological Supply Company/Printed in USA.

This kit addresses the following National Science Content Standards for grades 9–12:

Science as Inquiry

• Abilities necessary to do scientific inquiry

• Understandings about scientific inquiry

Life Science

• The molecular basis of heredity

• Biological evolution

Science and Technology

• Understandings about science and technology

Science in Personal and Social Perspective

• Natural and human-induced hazards

• Science and technology in local, national, and global challenges

History and Nature of Science

• Science as a human endeavor

Time Requirements

Preparation

Pour plates . . . . . . . . . . . . . . . . . . . . .1–10 days before lab . . .90 minutes

Copy student sheets . . . . . . . . . . . . . .3 days before lab . . . . . .15 minutes

Streak starter plates . . . . . . . . . . . . . .1–2 days before lab . . . .20 minutes

Set up student work areas . . . . . . . . .lab day 1 . . . . . . . . . . . .50 minutes

Procedure

Hand out Background and Pre- . . . . . .2 days before lab . . . . . .5 minuteslaboratory Inquiry (homework)

Pre-laboratory Inquiry Activity . . . . . .1 day before lab . . . . . .50 minutes

Lab: Transformation . . . . . . . . . . . . . .lab day 1 . . . . . . . . . . . .50 minutes

Analysis and discussion . . . . . . . . . . .lab day 2 . . . . . . . . . . . .50 minutes

Teacher’s Manual Transformation Kit for AP Biology

NOTES


Materials

Included in the kit: 8 Station 1 Station

transformation tubes 16 2

pack of glass beads 2 1

disposable inoculating loop 28 4

1-mL sterile transfer pipets 56 7

nichrome wire inoculating loop 1 1

sterile petri dishes 60 -

400-mL bottle of sterile LB agar 3 -

sterile 50 mM CaCl2, 3 mL 8 1

sterile LB broth, 3 mL 8 1

Teacher’s Manual and Student Guide 1 1

Order Form* for perishable materials— 1 1

4 mL ampicillin, 10 mg/mL 1 -

4 mL kanamycin, 10 mg/mL 1 -

LB plates - 3

LB/kanamycin plates - 2

LB/ampicillin plates - 2

200 µL, pGREEN plasmid, 0.005 µg/µL 1 1

200 µL, pAMP plasmid, 0.005 µg/µL 1 1

200 µL, pKAN plasmid, 0.005 µg/µL 1 1

MM294 slant culture 1 1

Needed, but not supplied:

containers with crushed ice

water bath or other method for holding water at 42°C

37°C incubator (optional)

microwave or boiling water bath

Bunsen burner or other device for flaming a loop

waste containers

thermometer

household bleach or ethanol

*If the kit with perishables is ordered, there will be no Order Form; instead,those items will be shipped at the same time as the other kit componentsand should immediately be treated as described below in the Storage andShelf Life section. Otherwise, follow the instructions on your Order Form torequest a suitable arrival date for your perishable items. Place your order atleast 2 weeks before your selected delivery date.

Storage and Shelf Life

Refrigerate the MM294 (E. coli) slant culture upon arrival. It is best used within 2 weeks but may be used up to 4 weeks after arrival.

Transformation Kit for AP Biology Teacher’s Manual

NOTES


Freeze or refrigerate the plasmid and antibiotics upon arrival. The plasmid is goodfor a year in the refrigerator, and for several years if frozen. The antibiotics aregood for a month in the refrigerator or 6 months in the freezer.

All other materials can be stored at room temperature. If kept sterile, the LB brothand CaCl2 are good for at least a year.

Safety Ensure that students understand and adhere to safe laboratory practices whenperforming any activity in the classroom or lab. Demonstrate the protocol forcorrectly using the instruments and materials necessary to complete the activities,and emphasize the importance of proper usage. Use personal protectiveequipment such as safety glasses or goggles, gloves, and aprons whenappropriate. Model proper laboratory safety practices for your students andrequire them to adhere to all laboratory safety rules.

When heating and pouring the Melt-n-Pour agar, use heat-resistant gloves. Ifheating in a microwave, once the agar begins to boil swirl the bottle every minuteor so to prevent boiling over. If the agar boils over and the resulting hot agar orsteam comes in contact with skin, it can cause serious burns.

Handling E. coli

The bacterial strain used in this laboratory is Escherichia coli, the same bacteriumused in most molecular biology labs and in educational labs of this nature. Sincesome strains of E. coli are associated with disease, safety concerns may arise.

Many naturally occurring strains of E. coli inhabit the gut of various animals,including cattle, swine, and humans. Some genetic variants of E. coli do causedisease. These variants contain disease-causing genes (e.g., those that code fortoxins causing intestinal upset). The laboratory strain supplied with this kit is aweakened version of the normal E. coli of the gut and does not contain thesedisease-causing genes. The strain is harmless under normal conditions. If introducedinto a cut or an eye, laboratory strains might conceivably cause an infection, so thefollowing standard safety precautions are recommended in handling the organisms.

Safety Tips for Handling E. coli

1. Always reflame the inoculating loop or cell spreader one final time beforesetting it down on the lab bench.

2. When pipetting suspension cultures, keep your nose and mouth away fromthe tip of the pipet to avoid inhaling any aerosol that might be created.

3. Avoid incubating plates longer than is necessary; longer incubation promotesthe growth of contaminating organisms.

4. Wipe down the lab bench with a 1:9 solution of household bleach to water(volume to volume) or with ethanol at the end of laboratory sessions.

5. Wash your hands before leaving the laboratory.

6. Keep waste containers at each student station for disposal of all materialsthat have come into contact with E. coli. This helps prevent contaminateditems’ being placed on the bench.


NOTES


7. Treat E. coli cultures, as well as tubes, pipets, transfer loops, and all othermaterials that have come into contact with the cultures in one of two ways:

a. Submerge them in 1:9 bleach-water solution for at least an hour. Thenplace the materials in the regular trash. Make sure that all surfaces of thematerials are in contact with the bleach during their time soaking.

b. Autoclave at 121°C, 15 lb/in2 for 15 minutes. Then place the materials inthe regular trash.

Teaching InquirySince the National Research Council published the National Science EducationStandards in 1996, the inquiry approach to science education has becomerecognized as a method that actively engages students in a learning process thatresults in greater mastery of scientific concepts. The findings of the NationalResearch Council support the evidence that an inquiry approach to educationhelps students gain an in-depth understanding of science by building uponprevious knowledge (Inquiry and the National Science Education Standards 2000).Students become empowered, taking responsibility for their learning byconducting inquiry investigations and communicating their discoveries.

Inquiry activities encourage students to explore questions in a scientific way. Instructured inquiry, the question is supplied to the student. A systematic procedurefor exploring the question and reaching a conclusion may also be provided. Inopen inquiry, the student directs the entire scientific investigation, determiningthe question to explore, the materials to use, the procedure to follow, and themethods used to analyze data.

This Lab's Approach

Carolina InvestigationsTM for AP Biology take a guided-inquiry approach to scienceinstruction. Generally, a teacher-guided activity is followed by an investigationplanned and carried out by student teams. Then, the teams present theirexperiments, findings, and conclusions. However, given the technical nature ofthe transformation procedure and the cost of the laboratory materials involved,this laboratory takes a different approach.

In this lab, you, as the instructor, will present students with a scenario and problemto be solved, a list of available materials, and the procedure for performingbacterial transformation. Students will design an experiment to solve the problem.Once each student group has planned their investigation, the class will discusseach group's experimental design. The discussion should focus on the conclusionsthat students expect to be able to make from their experiment and why certaincontrols must be included in order for definitive conclusions to be drawn.

Pre-laboratory questions test students’ prior knowledge. Big Idea Assessmentsadministered after the activities provide practice for the AP Biology exam’s free-response questions and help students internalize and effectively communicate thescientific concepts addressed by the investigation.


NOTES


Background InformationMost of the science content background has been included in the Student Guide.Read it to know what information your students have and have not been provided.Additional information has been included below to help you answer some questionsthat may arise during the lab or to provide a more in-depth background to yourstudents.

Antibiotics and Mechanisms of Antibiotic Resistance

Ampicillin

Ampicillin is a synthetic drug of the penicillin family of antibiotics. Interestingly,penicillin, the first antibiotic to be isolated, came from a soil fungus (Penicillium sp.). Itis believed that the fungus’s ability to produce penicillin evolved to help it compete withsoil bacteria. Ampicillin and other members of the penicillin family interfere with theformation of bacterial cell walls, thus preventing bacteria from growing and replicating.These antibiotics contain a chemical group called a beta-lactam ring. Ampicillin-resistant bacteria destroy the antibiotic by cleaving the ring, using an enzyme calledbeta lactamase. Beta lactamase is coded for by the ampicillin-resistance gene.

Kanamycin

Kanamycin is a member of the aminoglycoside antibiotic family. Kanamycinhinders the functioning of the 30S subunit of the prokaryotic ribosome and thus,interferes with protein translation. Kanamycin resistance genes code for enzymesthat inactivate kanamycin by adding a chemical group to the antibiotic.

Satellite Colonies

Under certain conditions, you may see satellitecolonies, small colonies that grow up around theampicillin-resistant colonies on your ampicillinplates. Around the ampicillin-resistant colonies,some ampicillin-sensitive bacteria may remain livingbut unable to replicate in the presence of theantibiotic. As the beta-lactamase secreted by aresistant colony diffuses further into thesurrounding agar, it destroys the ampicillin thereand allows the non-resistant bacteria to formcolonies. The older the ampicillin plates, or thelonger the plates are incubated, the greater thepossibility of getting satellite colonies.

References

Chain, E., H.W. Florey, A.D. Gardner, N.G. Heatley, M.A. Jenning, J. Orr-Ewing,A.G. Sanders. 1940. Penicillin as a therapeutic agent. Lancet Vol. ii: 226–8.

Fleming, A. 1929. On the antibacterial action of cultures of a Penicillium, withspecial reference to their use in the isolation of B. influenzae. The British Journalof Experimental Pathology Vol. 10, 226–36.

Walsh, C. 2000. Molecular mechanisms that confer antibacterial drug resistance.Nature Vol. 406: 775–81.


NOTES

Satellite colonies aroundampicillin-resistant coloniescontaining the pBLU® plasmid


Student Prior Knowledge

Before beginning this activity, students should be familiar with sterile technique. Ifthey are not already familiar with sterile technique, or if they need a review, use thebackground sheet in the appendix of the Student Guide for review or instruction.

Students should also be familiar with ampicillin and kanamycin and how they function.Background regarding this has been included in the teacher's background section.

Preparation1. Pour plates (1–10 days prior to lab).

Note: If you ordered the 1-station kit, the plates came pre-poured; skip tostep 2.

General points

• The plates should be prepared no more than 10 days before the lab. Workin a clean, quiet area, away from drafts. Wipe down your work surfacewith 70% ethanol or bleach before pouring the plates.

• You will pour 20 LB plates (16 for plating the transformed bacteria andcontrols, 4 for starter plates), 16 LB/amp plates, and 16 LB/kan plates.Label your petri dishes accordingly. Label the bottom of the plate aroundthe edge. This provides a clear view of the label without obscuring what ison the plate. There will be 8 extra petri dishes.

• Loosen the cap on the bottle of agar and melt the agar before pouringit, either in a boiling water bath or in a microwave oven. When heatingthe agar, be sure to wear a heat-resistant glove. Caution: Be extremelycareful when heating agar in a microwave as it can easily boil over andmay cause serious burns if it immediately comes in contact with your skin.

• When using a boiling water bath, make sure that the water in the bath orbeaker remains at or above the level of the agar in the bottle, and thatthe water remains at a full boil the entire time. Once the agar has beenplaced in the boiling water, it will take 45 to 55 minutes to melt. Scheduleadditional time for initially bringing the water to a boil.

• With microwave ovens, the time for agar to melt varies greatly, dependingupon an oven’s power. Allow at least 45 minutes. Caution: Keep a closeeye on the bottle once the agar begins to melt, and then swirl the bottleevery minute or so to prevent it from boiling over. Heat the agar until it iscompletely melted; there should be no lumps left in the bottle.

• Allow the agar to cool to 55°C (until the bottle can be held in a bare handwithout pain; it will still feel very warm). Agar that is too hot will inactivateany antibiotic that is added. After adding antibiotic, swirl the agar gently tothoroughly mix the antibiotic into the agar. Wait for any resulting bubbles topop, but do not allow the agar to get too cool for pouring.

• When pouring the plates, add just enough agar to cover the bottom ofthe dish approximately 5 mm deep. Remove the lid just enough to pourthe agar, and quickly replace the lid once the plate is poured.


NOTES


• After the agar has solidified, place the plates in the original sleeve or in a resealable bag and store them in the refrigerator. If there is a lot ofcondensation on the lids, allow the plates to sit out overnight to dry. Do not allow them to sit out any longer than one night.

Specific instructions

a. Use one bottle of LB agar to pour 18 LB plates. Do not add any antibioticto this bottle of LB agar.

b. Use the second bottle of LB agar to pour 2 LB plates and 16 LB/ampplates. After the agar has cooled, but before adding the antibiotic, pour 2LB plates. Then add the entire 4-mL vial of ampicillin to the remainingvolume in the bottle. Pour 16 LB/amp plates. The concentration of theampicillin in the vial is 10 mg/mL. The final concentration of ampicillin inthe plates will be approximately 100 µg/mL.

c. Use the last bottle of LB agar to pour the 16 LB/kanamycin plates. Afterthe agar has cooled, add the entire 4-mL vial of kanamycin to the 400 mLbottle. Pour 16 LB/kanamycin plates. The concentration of kanamycin inthe vial is 10 mg/mL. The final concentration of kanamycin in the plateswill be 100 µg/mL

2. Copy the Student Guide.

Copy the Pre-laboratory Inquiry and the Background in the Student Guide foreach student. If your students need additional instruction in sterile technique,make a copy of the Sterile Technique sheet in the Appendix of the StudentGuide for each student. Tables for collecting and organizing the data from thelab have also been included in the Appendix. Students are expected to devisetheir own ways of presenting their data; however, these tables are availableshould you wish to use them.

3. Streak the starter plates for the transformation lab.

General points

• If you plan to incubate the plates at 37°C, streak the plates 12–20 hoursbefore performing the transformation. If you plan to incubate at roomtemperature, then streak the plates 24–40 hours before performing the transformation.

• Use sterile technique when streaking plates. (You may wish to review theinstructions on sterile technique in the Appendix.)

• The goal in streaking starter plates is to obtain single isolated colonies for usein the transformation. This is important for the success of the experiment. Donot use bacterial cells from an overgrown area of the plate, and do notexceed the recommended incubation times. The bacteria need to be inthe exponential growth phase in order for the transformation to work.

• For the 1-station kit, you will need one starter plate. For the 8-station kit,you will need four starter plates (one plate will be shared between twostudent groups). Use LB plates to make the starter plates.


NOTES


Specific instructions

a. Label the LB plates with the date and the name of the bacteria, E. coli(MM294.)

b. Hold the wire inoculating loop like a pencil and sterilize the circle at theend using the Bunsen burner or other flame until it glows red. Do thesame with the lower third of the wire next to the loop. Do not set theloop down.

c. With the other hand, grasp the slant culture of E. coli between yourthumb and two fingers. Remove the vial cap using the little finger of thehand holding the inoculating loop. Avoid touching the rim of the cap.Quickly pass the mouth of the slant through the flame.

d. Stab the inoculating loop into the side of the agar to cool it. Then dragthe loop across an area of the E. coli culture where bacterial growth isapparent. Remove the loop, flame the vial mouth, replace the cap, and setthe vial down.

e. Inoculate the plate in four streaks as described below. The loop will beloaded with E. coli from the slant culture for the first streak only. Somepeople streak the plate while lifting the lid only high enough to admit theloop. Others place the lid on the sterile lab bench while streaking. Eitherway, lift or remove the lid only long enough to perform the streaking.

How to streak a plate

f. To prevent contaminating the lab bench, reflame the loop before setting it down.


NOTES

1 2

3 4

Glide the loop tip back and forth across one quadrant of the agar plate to makesome zigzags (1). Avoid gouging the agar. Replace the lid of the plate. Reflame theloop and cool it by stabbing it into the side of the agar away from the first(primary) streak. Draw the loop tip once through the primary streak and continuestreaking a zigzag across the agar surface (2). Reflame the loop and cool it in agaras before. Draw the loop tip once through the secondary streak, and makeanother zigzag streak (3). Reflame the loop and cool it. Draw the tip once throughthe tertiary streak, and then make the final zigzag streak (4).


g. Repeat steps b–e for any remaining starter plates.

h. Place the starter plates upside down in a 37°C incubator for 12–20 hours.Alternatively, grow them at room temperature for 24–40 hours. Remember:Do not exceed the recommended incubation times. The plates areturned upside down to prevent any condensation that might collect on thelids from falling on the agar and causing colonies to run together.

4. Set up for lab day.

The night before the lab:

a. Pre-warm incubator to 37°C (if you are using one).

b. Chill the vials of CaCl2 in the refrigerator.

c. Turn on a 42°C water bath. (If the lab is late in the day, do this themorning of the lab.)

Note: If you do not have a water bath, a small disposable cooler filledwith 42°C tap water will suffice. Make sure that you have the water closeto temperature just prior to the lab and make any final adjustments usinghot or cold water during the 15-minute incubation that precedes the heatshock. If you use this method, you will set it up on the day of the lab.

The day of the lab:

Set up 1 or 8 student lab stations (depending on the kit you are using), eachwith the following materials:

2 kanamycin plates

2 ampicillin plates

2 LB plates

2 transformation tubes

3 disposable inoculating loops

1 vial calcium chloride (chilled and on ice)

1 vial LB broth

1 waste container for items that come into contact with E. coli(e.g., 1-L plastic beakers)

1 container of crushed ice (e.g., plastic beaker)

1 permanent marker

1 tube rack to hold transformation tubes

1 container for used glass beads (e.g., a large beaker or a wide-mouthed resealable plastic container)

Place the following at a shared station:

starter plates (between two groups)

glass beads for spreading

plasmids 1, 2, and 3


NOTES


Procedure

Pre-laboratory Inquiry

1. Divide your students into eight groups. If you are using the 1-station kit,students will work as one group.

2. Before the day you plan to have them perform the inquiry, provide eachstudent with the Background and the Pre-laboratory Inquiry portions of theStudent Guide. As homework, have students read the Background andanswer the questions.

The Background contains some history on the discovery of naturally occurringtransformation and its importance. It also provides background on plasmids andtheir role in antibiotic resistance and how transformation and plasmids are nowused as tools for genetic engineering. Information on antibiotic resistance as amodel of natural selection is also included.

The Pre-laboratory Inquiry explains the problem that students are to solve,some reminders to help guide their thinking, some pre-activity questions, anda list of available materials.

3. Make sure that your students understand that the Pre-laboratory Inquiry Activityis a precursor to the lab, and that no actual materials will be used at this point.

4. Discuss the answers to the questions in the Pre-laboratory Inquiry and makesure that the students understand the answers before they move on.

5. Working in their assigned groups, have students design an experiment toaddress the challenge posed in the Pre-laboratory Inquiry. Tell them thatduring the lab the class may have to work together or compile results to getall the data that they need. If any teams need help with their planning, askquestions that encourage critical thinking, and remind students aboutappropriate experimental controls. Once each group has developed anexperimental design, have the groups present their designs to the class forcritiquing. At the end of the class discussion, students should understand theneed for the controls included in the transformation procedure.

Bear in mind the following regarding the controls:

• Bacterial cells with no plasmid added to them should be treated in thesame way as the transformed cells. This control demonstrates that thephenotypic change seen in the bacteria following the transformation is aresult of the introduction of the plasmid DNA.

• Both the transformed and mock-transformed bacteria (i.e., those withoutplasmid added) should be plated on LB without antibiotics to demonstratethat the bacteria are viable following the transformation procedure.

• Plating the transformation on both plain LB and LB/amp plates alsodemonstrates that only a fraction of the cells take up the plasmid.

The ideal way to set up the experiment is illustrated in the TransformationLaboratory Procedure in the Student Guide, where students are instructedhow to label their plates.


NOTES


Lab

1. After the Pre-Laboratory Inquiry Activity but before the transformationactivity, distribute the Transformation Laboratory Procedure to each studentso that they can become familiar with the procedure.

2. Assign each group a plasmid.

3. Have students perform the transformation as described in their instructions.Distribute the glass beads to the students after step 13, during the roomtemperature incubation in LB. Have students add approximately four beadsto each plate. Remind them to add the beads with the plates upside down(lid side on the bench) as the beads are less likely to bounce off the plastic lidthan they are the agar.

4. Have students answer the Laboratory Questions.

5. Administer the Big Idea Assessments to test student understanding of theconcepts addressed in the laboratory and background material.

Answers to Questions in the Student Guide

Questions from the Pre-laboratory Inquiry

1. What is a plasmid?

A plasmid is a small, usually circular, extra-chromosomal piece of DNAthat exists in nature in some bacteria and yeasts. They can betransferred between organisms. In the laboratory, they are used tomanipulate and introduce DNA of interest into a bacterium, and area powerful tool in genetic engineering.

2. What is transformation?

Transformation is the uptake of exogenous, naked DNA by a cell.The newly adopted DNA can become a heritable part of the cell'sgenetic material.

3. Why is naturally occurring transformation beneficial to bacteria?

Naturally occurring transformation is beneficial to bacteria, because itprovides them with access to a broader array of genetic material,which increases their chances of being able to adapt to theenvironment.

4. Why is transformation useful to research scientists?

Transformation is useful to research scientists because it enables themto introduce foreign DNA into bacteria. This allows them to furtherstudy or work with gene(s) on the plasmid DNA and the protein(s)that the gene(s) code(s) for. (In some cases, introducing the plasmidalso enables researchers to manipulate and study genes alreadypresent in the bacterial genome.)

5. Should you plate some of your transformed bacteria onto plates withantibiotics? Why or why not?

Yes, this ensures that those bacteria that take up the plasmid willretain it and allows you to select for those bacteria that have actuallytaken up the plasmid.


NOTES


6. What would you expect to see if you plated some of your transformedbacteria onto a plate without antibiotic? Would there be an advantage todoing this (in terms of understanding your results)? Explain.

You would expect to see a lawn. Yes, this would allow you to assess cellviability. How viable the cells are is important to know in the event thatthere are no colonies on the plate that does contain antibiotic. Inaddition, being able to directly contrast the lawn on the antibiotic-freeplate with the colonies present on the plate with antibiotic is a gooddemonstration of how small a percentage of the cells actually took upthe plasmid. The contrast also indicates that the antibiotic is good(antibiotics can deteriorate from long or improper storage).

7. To transform bacteria with plasmids, technicians first make the bacteriacompetent (capable of taking up DNA) by placing them in calcium chlorideand chilling them. Plasmid is then added to the competent bacteria and theplasmid/bacteria combination is taken through a few more steps to makethe bacteria take up the DNA. In your experiment, should you treat a tubeof bacteria that you do not add plasmid to exactly as you do the tube ofbacteria that you will transform? Why or why not?

Treating bacteria that you have not added plasmid to exactly as you dothe bacteria that you add plasmid to, provides a control demonstratingthat the antibiotic-resistant colonies that appear on the plate are aresult of the plasmid being taken up by the cells (i.e., that a change ingenotype causes a change in phenotype). In the absence of plasmid,there should be no antibiotic-resistant colonies present on the plate.

Laboratory Questions

The identity of the plasmids that the students are using is as follows:

Plasmid 1: pAMP (contains a gene for ampicillin resistance)

Plasmid 2: pKAN (contains a gene for kanamycin resistance)

Plasmid 3: pGREEN (contains genes for ampicillin resistance and green fluorescentprotein)

1. Record your results and conclusions using the organizational method youdevised in the Pre-laboratory Inquiry Activity.

The expected results are displayed below in the form of the optional,reproducible data tables included in the Appendix. Student's datamay be organized differently, but students do need to collect thedata indicated and to have a methodical way of recording theirconclusions. The number of colonies students see on their platesshould be in the range of the numbers shown in this sample table.Occasionally, if the procedure has not been closely followed, or ifstudents are particularly skilled, the number of colonies seen will bebelow or above these ranges.


NOTES

+ plasmid – plasmid + plasmid – plasmid + plasmid – plasmid Color of colonies(where they appear)

Plasmid 1 25–100colonies 0 colonies 0 colonies 0 colonies lawn lawn yellow

Plasmid 2 0 colonies 0 colonies 25–100colonies 0 colonies lawn lawn yellow

Plasmid 3 25–100colonies 0 colonies 0 colonies 0 colonies lawn lawn yellow/green or green

LB AMPICILLIN PLATES LB KANAMYCIN PLATES LB PLATES


2. Did you observe what you expected to? If not, how would you explain yourobservations?

If students see no colonies on any of their plates, the most commonsources of error include

• incubating the starter plates for too long.• not moving the tubes directly from ice to the water bath

and back to the ice again during the heat shock.• not getting DNA onto the inoculating loop.

If student's colonies are not appropriately green, let the plates sit outat room temperature for a day or two. Occasionally, the greenphenotype takes a few days to appear.For other circumstances that may cause anomalous results, see theanswers to question 3.

3. The results from three different experiments are as described in a, b, and c.Something has gone wrong with each of these experiments. Use thecontrols to figure out what has gone wrong in each experiment.

a. Results from Experiment 1

Plate ResultLB/amp+plasmid plate (no lawn, no colonies)LB/amp–plasmid plate (no lawn, no colonies)LB/kan+plasmid plate (no lawn, no colonies)LB/kan–plasmid plate (no lawn, no colonies)LB+plasmid plate (no lawn, no colonies)LB–plasmid plate (no lawn, no colonies)

There should be a lawn on both of the LB plates. The absence of the lawn indicates that the bacteria were not viable.

b. Results from Experiment 2

Plate ResultLB/amp+plasmid plate (lawn)LB/amp–plasmid plate (lawn)LB/kan+plasmid plate (clean plate)LB/kan–plasmid plate (clean plate)LB+plasmid plate (lawn)LB–plasmid plate (lawn)

The LB/amp–plasmid plate should have no bacteria on it, and either the LB/amp+plasmid plate or the LB/kan+plasmid plate should have colonies on it. The observation that both ampicillin plates have lawns on them indicates that the ampicillin was not good or that the entire culture used to start the experiment was


NOTES

Does the plasmid belowcontain

an ampicillin-resistancegene?

a kanamycin-resistancegene?

a green fluorescentprotein gene?

Plasmid 1 yes no no

Plasmid 2 no yes no

Plasmid 3 yes no yes


ampicillin-resistant. That the MM294s were ampicillin-resistant is unlikely. The most likely explanation is that somehow the antibiotic has been inactivated. This can happen if the antibiotic was added when the LB agar was too hot, or if the antibiotic was too old, or not stored properly.

c. Results from Experiment 3

Plate ResultLB/amp+plasmid plate (colonies)LB/amp–plasmid plate (colonies)LB/kan+plasmid plate (clean plate)LB/kan–plasmid plate (clean plate)LB+plasmid plate (lawn)LB–plasmid plate (lawn)

The observation that there were colonies on the LB/amp–plasmid plate indicates that there were some ampicillin-resistant colonies contaminating the transformation. If the number of colonies on the +plasmid and –plasmid plates was the same, it is likely that thetransformation did not work. A much larger number of colonies on the +plasmid plate indicates that the transformation probably worked but that some of the colonies are not a result of the transformation.

4. Having a way to measure transformation efficiency helps in discussingresults or in comparing transformations that were not done at the sametime. Transformation efficiency is expressed as the number of transformedcolonies (in this case those that are antibiotic-resistant) per microgram ofplasmid used in the transformation.

a. Figure out how you would calculate transformation efficiency(i.e., number of colonies/µg of plasmid used). You used 10 µL of plasmid at a concentration of 0.005 µg/µL.

The mass of plasmid used: 10 µL × 0.005 µg/µL = 0.05 µgThe total volume of the cell suspension prepared: 250 µL (CaCl2) + 250 µL (LB) + 10 µL (plasmid) = 510 µL

If you spread 100 µL on the plate, the fraction of the total transformation volume spread: 100 µL/510 µL = 0.196The total mass of the plasmid in the cell suspension spread: 0.05 µg × 0.196 = 0.0098 µgThe number of colonies observed/0.0098 µg = transformation efficiency.

b. Now use the method you devised above to determine the transformationefficiency for the transformation performed by your group.

Answers will vary. Given the amount of DNA used, for 100 coloniesthe transformation efficiency would be 10,204 colonies/µg.

c. What might be sources of error in calculating this number?

The greatest sources of error will result from students’ measurement of 250 µL and 10 µL. Some error may result from error in measuring and some from the limited precision of the measuring devices used.Another source of error is how accurately the colonies were counted.

5. You are making ampicillin plates. Before pouring the plates, you add 2 mL of10 mg/mL to the 400-mL bottle of LB agar that you use to pour the plates.


NOTES


What is the final concentration of the ampicillin in the plates? Express youranswer as µg/mL. Show your work.

C1V1 = C2V2

10 mg × 2 mL =

? mg × 400 mL

mL mL

20 mg =? mg

× 400 mL (Divide both sides by 400 mL.)mL

0.05 mg =

? mgmL mL

0.05 mg ×

1000 µg =

? mgmL mg mL

? =50 mgmL

6. Again, you are making ampicillin plates using LB agar. You are given a vial ofampicillin that is labeled as a 1% solution and told that you need to make 40 plates using this solution. Assuming that you will need 25 mL per plate,what volume of LB agar solution should you make to prepare the 40 plates?What volume of the 1% ampicillin solution do you need to add to this volume ofLB agar if the final concentration of ampicillin in the plates should be 50 µg/mL?Hint: Percentage is an expression of weight of solute per volume of solution.

40 plates × 25 mL/plate = 1000 mLA 1% solution means that it contains 1 g of ampicillin per 100 mL.Convert this to µg/mL to make the calculation easier.

1 g×

106 µg =

10,000 µg100 mL g mL

Then use C1V1 = C2V2 to find the volume of ampicillin (V1) needed.

10,000 µg × V1 =

50 µg × 1000 mL Solve for V1.

mL mLV1 = 5 mL

You should use 5 mL of the 1% ampicillin stock.

Big Idea Assessments

1. Give a specific example of how the introduction of a gene into a bacteriumcan change the phenotype of the bacterium. Also explain the specific role ofthe protein expressed by the gene in changing the bacterium's phenotype.

Any of the following may be used. Students may have also foundother examples.Introduction of an ampicillin-resistance gene into a bacterium canchange the bacterium from being ampicillin sensitive to ampicillinresistant. The protein expressed by the gene is beta-lactamase; thisenzyme cleaves the ampicillin, making it ineffective. Introduction of a kanamycin-resistance gene into a bacterium canchange the bacterium from being kanamycin sensitive to kanamycinresistant. The protein expressed by the gene is an enzyme thatinactivates kanamycin by adding a chemical group to it.Introduction of the GFP gene will turn the colonies green. The geneexpresses a protein whose structure makes this possible.

2. Griffin performed experiments demonstrating that when live,nonpathogenic S. pneumoniae (which produce rough-surfaced colonies) are


NOTES


mixed with killed smooth-surfaced S. pneumoniae (which are pathogenicwhen alive) and then injected into mice, the mice become ill. Bacteriaisolated from these sick mice form the smooth colonies characteristic of thepathogenic strain. What happened to the bacteria to make thempathogenic to the mice?

The live, rough-appearing bacteria were transformed by DNA fromthe dead, smooth-appearing bacteria. Taking up this DNA enabledthe bacteria to make the polysaccharide coating that helps themevade the mouse's immune system.

3. Briefly describe how the experiments of Avery, McCarty, and MacLeod,building on the work of Griffith, demonstrated that DNA was the moleculethat passed on traits.

Their experiments demonstrated that purified DNA from the deadsmooth-appearing cells was able to transform the rough-appearingcells, so that they now formed smooth-appearing colonies. They alsodemonstrated that destroying the DNA with enzymes destroyed thetransforming principle.

4. You have isolated one of the genes for producing one of the blood-clottingproteins needed by some hemophiliacs. Briefly describe how you couldcreate bacteria that would produce this protein.

The isolated gene could be inserted into a plasmid containing anantibiotic-resistance gene. The plasmid containing the gene couldthen be transformed into bacteria that would be grown in thepresence of the appropriate antibiotic.

5. Name the two ways in which bacteria can acquire new genetic material.Both ways are examples of lateral (or horizontal) gene transfer.

Transformation and conjugation.

6. Assume that you transform bacteria with a plasmid containing an ampicillin-resistance gene. Instead of directly plating the transformed population asyou did in this lab, you set up two liquid cultures of them, one that containsampicillin and one that does not. You will then assay these cultures onplates at two different times: immediately after you set up the cultures, andthen again after the bacteria have been in culture for an extended period.The assays will demonstrate the number of ampicillin-resistant vs. ampicillin-sensitive bacteria in each culture at each time. To perform each of the twoassays, you prepare serial dilutions of the two cultures and plate them ontoLB plates with and without ampicillin (the dilution is simply to ensure thatyou will get some plates on which you can distinguish separate colonies).

Describe what you expect to observe in the initial assay and in the secondassay. What, if any, differences might you expect in terms of the ratios ofampicillin-resistant and nonresistant bacteria?

Initially, there should be more colonies on the LB plate than on theLB/amp plate with respect to both cultures. This is because only asmall proportion of bacteria are actually transformed and theantibiotic has not had any time to exert selective pressure.

Once the cultures have been allowed to grow for an extendedperiod, the relative number of ampicillin-sensitive vs. ampicillin-resistant colonies changes, because of the selective pressure exertedby the antibiotic. The culture grown in the presence of antibioticshould produce just as many colonies on the LB plate as on theLB/amp plate. All of the colonies on both plates should be ampicillin


NOTES


resistant. The culture grown in the absence of antibiotic shouldproduce no colonies on the LB/amp plate because any ampicillin-resistant bacteria will have been outcompeted over time by the moreabundant nonresistant bacteria. There should be plenty of colonieson the plain LB plate.

Helpful Hints1. Do not overincubate the starter plates.

2. Emphasize the following to your students:

a. When scraping bacterial cells from the plates, do not scrape any agar along with the cells. The agar may inhibit the transformation.

b. Make sure the cell mass scraped from the plate drops off into the calcium chloride and is not smeared on the side of the tube.

c. Resuspend the cells immediately after they are placed in calcium chloride as they become difficult to resuspend if they are left in the solution for any length of time. Thorough resuspension of the cells is important.

d. During the heat shock in the transformation procedure, make sure that the bacteria being transformed are on ice when carried to the 42°C water bath and that the tubes are placed directly back into the ice from the water bath.

3. As supplied, the glass beads are sufficiently sterile that if the recommendedincubation times are not exceeded, they can be used as is. If you reuse thebeads or are especially concerned with sterility, they can be sterilized byautoclaving, boiling for 10 minutes, or washing in ethanol or a 10% bleachsolution for 10 minutes.

4. If the incubation on ice in step 10 and the incubation at room temperature inLB exceed 15 minutes, this will not adversely affect the transformation.However, do not let either incubation exceed 30 minutes. Do not alter any ofthe other experimental parameters.

Extension ActivitiesIf you would like students to learn more about antibiotic resistance and explore agood model of evolution, you may wish to present them with the followingchallenge. They have access to some of the transformed bacteria left in thetransformation tubes they did their transformation in. Provide them with LB broth,kanamycin plates, ampicillin plates, LB plates, LB medium, kanamycin, ampicillin,sterile culture tubes or flasks, sterile loops for inoculation, sterile bulb pipets fortransferring E. coli cultures to plates, and some way to spread bacteria cultures onthe plates. Note that these supplies for this extension activity are not includedwith this kit and will have to be purchased separately. Ask students to come upwith an experiment demonstrating that, over time, a bacterial population canchange its phenotype with respect to antibiotic resistance. Have students write a detailed protocol in which they clearly state

1. what they are testing.

2. what they think will happen and why.


NOTES


3. the materials they are going to use.

4. the steps they will take to perform the experiment (i.e., a detailed procedure).

5. the form of data collection and presentation.

One possible experiment follows:

Use the remaining transformed cells that they have not plated to start two 2-mL liquidcultures—one with antibiotic and one without. Subculture the bacteria (transfer asmall volume of the culture) to a tube of fresh medium with or without antibiotic (as is appropriate) every 24 hours. The bacterial population should shift its phenotypein approximately 2 days. The small fraction of those cells that were transformed willbe antibiotic resistant and will quickly (within a day or two) come to dominate a population that is grown in the presence of antibiotic. In the absence of antibiotic,the small number of antibiotic-resistant bacterial cells will be quickly outgrown andoutcompeted by the nonresistant (i.e., normal) strain.

Students can assay the bacterial population in multiple ways, but the most precisemethod would be for them to make serial dilutions of the bacteria and then toplate the dilutions most likely to allow them to count individual colonies on LBplates with and without antibiotic. As a guideline—if you plate 100 µL of thedilution, a saturated culture of bacteria will need to be diluted approximately 105

or 106 for students to be able to discern individual colonies. They should assay thecultures at various times to see how the ratio of antibiotic-resistant to nonresistantcolonies shifts. If they assay the culture immediately after it is set up, they willneed to use a lower serial dilution when plating.

Related ProductsFollowing is a list of related items available from Carolina Biological SupplyCompany. For more information, please refer to the most recent Carolina™Science catalog, call toll free 800-334-5551, or visit our Web site atwww.carolina.com.

RN216620 Ready-to-Pour Luria Broth Agar

RN216621 Ready-to-Pour Luria Broth Agar + ampicillin

RN216622 Ready-to-Pour Luria Broth Agar + kanamycin

RN216858 Ampicillin

RN216861 Kanamycin

RN216650 Luria Broth

RN215856 Disposable Inoculating Loops

RN215090 Transformation Tube, 15 mL

RN214551 1-mL Sterile Transfer Pipets


NOTES

NAME

DATE

©2014 Carolina Biological Supply Company/Printed in USA. S-1

CarolinaTM Transformation for AP Biology

Background

Student Guide

Transformation and Its Role in Discovering the Function of DNA

The discovery of transformation in bacteria was crucial in understanding that DNA is the material ofinheritance. Transformation is the uptake of exogenous, naked DNA by a cell. The newly adopted DNA canbecome a heritable part of the cell's genetic material. Transformation was discovered through work doneby several scientists, or groups of scientists, each of them building on the work of the others. In 1928, theEnglish scientist Frederick Griffith was studying the bacterium Streptococcus pneumoniae. This organismcauses pneumonia, which was a leading cause of death. Griffith worked with two strains of S. pneumoniae:one that caused disease and whose colonies were smooth in appearance, and another that did not causedisease and whose colonies were rough in appearance. The smooth appearance of the pathogenic form ofS. pneumoniae resulted from a specific type of external polysaccharide coating, which is now known to bethe mechanism by which the strain evades a host’s immune system.

Griffith's experiments involved injecting the mice with the two different strains of S. pneumoniae. When heinjected the smooth strain, the mice became ill and died. When he injected the rough strain, the mice stayedhealthy. However, when Griffith mixed heat-killed smooth cells (which caused no disease when injected intomice) with living rough cells (which caused no disease when injected into mice) and injected thatcombination into mice, surprisingly, the injected mice became ill as if they had been injected with live smoothcells. When Griffith isolated S. pneumoniae cells from the dead mice, the bacteria formed smooth colonies.Griffith concluded that the living rough cells had been transformed into smooth cells as the result of beingmixed with the dead smooth cells. Sixteen years later Avery, McCarty, and MacLeod showed that the"transforming principle," the substance from the heat-killed smooth strain that caused the transformation,was DNA. They did so by demonstrating that purified DNA from the killed smooth cells was able to transformthe live rough cells and that enzymes which destroyed DNA destroyed the "transforming principle."

Griffith’s transformation experiment with smooth and rough strains of Streptococcus pneumoniae and mice


Natural Transformation—an Example of Lateral (Horizontal) Transfer of Genetic Material

Some bacteria, including Streptococcus pneumoniae, undergo transformation naturally. Bacteria thattransform naturally have mechanisms for transporting the DNA into the bacterial cell. Once inside the cell,the base sequence of the new DNA is compared to the bacterium's DNA. If enough similarity in sequenceexists between the new DNA and part of the bacterium's existing DNA, the new DNA can be substituted forthe homologous (like) region of the bacterium's DNA. If the new DNA is not similar to the bacterium'sDNA, it is not incorporated into the genome and is broken down by intracellular enzymes.

Why would bacteria have mechanisms for taking up free DNA from the environment? Taking up free DNAfrom the environment increases the organism’s access to diverse genetic material and thus increases theability of the organism to adapt to the environment. For example, Neisseria gonorrhea, the causative agentof gonorrhea, takes up DNA from members of its own species. In doing so, it often takes up DNA thatallows it to alter the character of its surface proteins. Since our immune system makes antibodies to thesesurface proteins in order to fight infection by N. gonorrhea, if the organism changes the nature of thesesurface proteins, it is more likely to evade our immune system.

From this example it is easy to understand how lateral (also called horizontal) gene transfer, the transfer ofgenetic material between organisms that do not have a parent/offspring relationship, plays a role in howmicroorganisms adapt to their environment. (In contrast, transfer of genetic material from parent tooffspring is referred to as vertical gene transfer.) Naturally occurring transformation is one example ofhorizontal gene transfer. See the section on plasmids for information regarding conjugation, anothermechanism of horizontal gene transfer.

Artificial Transformation

Scientists took natural transformation and developed it into something they could use as a powerful tool inthe lab. It is relatively rare for most bacteria to take up DNA naturally from the environment. However, bysubjecting bacteria to specific artificial conditions, scientists are able to cause bacteria to consistently takeup DNA. Bacteria that are able to take up DNA are referred to as competent. Making cells in the labcompetent usually involves changing the ionic strength of their medium and subjecting them to severaltemperature changes, one of which involves heating the cells in the presence of positive ions (usuallycalcium). Another method for making cells take up DNA at an artificially high rate involves exposing themto high voltage. This method is called electroporation.

Once DNA is taken into a host bacterial cell, the genes coded for by the DNA need to be expressed (i.e.,transcribed and translated). In addition, the DNA needs to persist in the cell. However, as discussed above,if transformed DNA is to persist in the cell, it must be integrated into the genome, and in order tointegrate into the genome it must be similar in sequence to at least some part of the organism's DNA.Often, when researchers want to introduce DNA into a bacterial cell, they want to introduce DNA that isquite different from that of the existing bacterial genome. To ensure that the introduced DNA will persistin the cell, scientists insert the DNA of interest into something called a plasmid and then transform thebacterial cells with the plasmid DNA.

Plasmids

Plasmids are small, usually circular pieces of extra-chromosomal DNA that exist in nature in bacteria andyeasts and are sometimes transferred between individuals. Bacteria transfer them in a process called"conjugation," in which there is physical contact between the two cells. Plasmids have been harnessed andmodified to serve a pivotal role in molecular biology. Each plasmid has an origin of replication (a sequenceof bases at which DNA replication begins). Because of this origin of replication, plasmid DNA (unlike

Transformation Kit for AP Biology Student Guide


fragments of transformed DNA that are not integrated into the organism's genome) can replicate in thehost bacterial cell. In addition, each daughter cell receives copies of the plasmid upon cell division. Thus,DNA inserted into a plasmid introduced into a bacterial cell can persist in the cell and in the cell's offspring.Because of this, plasmids provide an ideal mechanism for introducing foreign DNA into bacterial cells andwere critical in the development of genetic engineering.

Selective Markers

There is one more key step involved in making transformation work in the lab. In the preceding paragraph,it was stated that DNA introduced into a cell via a plasmid will persist in the cell and its offspring. This istrue, but there is a catch. Bacteria, in nature and in a crowded culture flask, are in intense competition forsurvival. Plasmids do not always contain genes that are beneficial for survival, and they do take extraenergy to maintain. The tendency is for bacteria to lose their plasmids unless there is an advantage toretaining them. One way to force a bacterium to keep a plasmid is to maintain the bacterium in thepresence of an antibiotic that will kill it unless it develops resistance to the antibiotic. Consequently,researchers grow transformed bacteria in the presence of antibiotic and include the appropriate antibiotic-resistance gene along with the other desired DNA on the plasmid that they want the bacteria to retain.The gene for antibiotic resistance is called a selective marker; not only does it force the bacteria to keepthe plasmid, it allows the researcher to select for those bacteria that have successfully taken up the plasmidof interest. Only those bacteria that have taken up the desired plasmid will survive.

The plasmids that you will work with in this lab activity use either ampicillin or kanamycin as a selectivemarker. You will use either ampicillin or kanamycin to select for bacteria that have actually taken up the plasmid.

Antibiotics, Antibiotic Resistance, and Evolution

This lab demonstrates one way in which bacteria can acquire antibiotic resistance—through the acquisitionof a plasmid with a gene for antibiotic resistance. In this lab, the bacteria were artificially induced to takeup a plasmid with an antibiotic resistance gene. However, remember that in nature plasmids are passedback and forth between bacteria, and that many naturally occurring plasmids contain antibiotic-resistancegenes as well.

While antibiotic resistance is a helpful tool in the lab, the development of antibiotic resistance in nature is aserious problem. The discovery of antibiotics is one of the most important medical breakthroughs; it hasallowed many people to survive what would have once been lethal bacterial infections. However theeffectiveness of many antibiotics has decreased over the years as bacteria have developed resistance to them.

A bacterial population's development of antibiotic resistance is an example of evolution at work. Throughrandom mutation (e.g., some bacteria have developed resistance through mutations in the gene coding forthe 30S ribosome) or possession of a plasmid with an antibiotic-resistance gene, bacterial populationsinclude a few individuals with genes providing resistance to one or more antibiotics. When the populationis exposed to an antibiotic, there is selective pressure in favor of the few individual bacteria that areresistant to the antibiotic. When these antibiotic-resistant bacteria divide, they pass the resistance trait onto their offspring. If the antibiotic persists in their environment, these offspring then come to dominateand eventually to constitute most, if not all, of the population.

The more prevalent the use of an antibiotic, the greater is the selective pressure in favor of antibioticresistance, and the more likely that bacterial populations evolve to become resistant. Some practices inanimal husbandry provide a good example of this dynamic. On many large farms, farmers add antibioticsto the animals’ feed to minimize infection among the animals, especially if they are grown in crowded



conditions. This sustained use of antibiotics contributes to the evolution of antibiotic-resistant bacteria.Another factor involved in increasing drug resistance in bacteria is doctors prescribing antibiotics topatients who do not really need them. Some doctors tend to prescribe or are pressured into prescribingantibiotics for patients who have viral infections, even though viruses are not killed by antibiotics.

To combat the problem of antibiotic resistance, researchers continually try to develop new antibiotics to killpathogenic bacteria in novel ways. To do this the researchers study bacteria to gain a thorough understandingof how they function.

Amabile-Cuevas, A.F. 2003. New antibiotics and new resistance: in many ways, the fight against antibioticresistance is already lost; preventing bacterial disease requires thoughtful new approaches. AmericanScientist Vol. 91(2):138–49.

Avery, O.T., C.M. MacLeod, and M. McCarty. 1944. Studies on the chemical nature of the substance inducingtransformation of pneumococcal types. J. Exp. Med. Vol. 79:137–58.

Cohen, S., A.C.Y. Chang, L. Hsu. 1972. Nonchromosomal antibiotic resistance in bacteria: genetictransformation of Escherichia coli by r-factor DNA. Proceedings of the National Academy of Science USA.Vol. 69(8):2110–4.

French, G.L. 2010. The continuing crisis in antibiotic resistance. International Journal of Antimicrobial Agents,Vol. 36S3:S3–S7.

Griffith, F. 1928. The significance of pneumococcal types. Journal of Hygiene, Cambridge, England Vol.27:113–59.

Levy, S.B. 1998. The challenge of antibiotic resistance. Scientific American Vol. 278(1):46–53.

Miller, R.V. 1998. Bacterial gene swapping in nature. Scientific American Vol. 278(1):66–71.

Ochman, H., J.G. Lawrence, E.A. Groisman. 2000. Lateral gene transfer and the nature of bacterial innovation.Nature Vol. 405:299–304.

Sparling, P.F., G.D. Biswas, T.E. Sox. 1977. Transformation of the gonococcus. 155–76 in The Gonococcus. R.B.Roberts ed., Wiley and Sons.

Walsh, C. 2000. Molecular mechanisms that confer antibacterial drug resistance. Nature Vol. 406:775–81.



Pre-Laboratory Inquiry

You work on a team in a research lab. You have three separate plasmids in your lab, but the labels havecome off of the tubes and you no longer know which plasmid is which. Come up with an experiment thatwill give you a definitive answer as to which plasmid is which. For the time being, you will refer to theplasmids as plasmids 1, 2, and 3. You know the following:

• One plasmid has a kanamycin-resistance gene.

• Two plasmids have an ampicillin-resistance gene.

• In addition to having an antibiotic-resistance marker, one plasmid also codes for the gene for greenfluorescent protein (GFP), a protein from the bioluminescent jellyfish Aequorea victoria.

Bacteria that produce green fluorescent protein look green under white light and fluoresce underultraviolet light.

Assume that each research team has access to the following materials and is responsible for identifying oneof the three plasmids. (Note: You will not actually use any materials during this pre-lab inquiry.) Beforeplanning your experiment, answer the questions below to ensure that you understand some of the basicconcepts involved in the lab. Some of these questions are specifically designed to help you think aboutsetting up your experiment.

starter plates (plates containing E. coli in the rapid, or exponential, stage of growth)

kanamycin plates

ampicillin plates

LB plates

plasmids 1, 2, and 3

transformation reagents, tubes, and equipment (inoculating loops, calcium chloride, LB broth, ice, 42°C water bath)

glass beads for spreading bacteria

1. What is a plasmid?

2. What is transformation?

3. Why is naturally occurring transformation beneficial to bacteria?

4. Why is transformation useful to research scientists?



Note: Questions 5–7 relate directly to the experiment you are planning.

5. Should you plate some of your transformed bacteria onto plates with antibiotics? Why or why not?

6. What would you expect to see if you plated some of your transformed bacteria onto a plate withoutantibiotic? Would there be an advantage to doing this (in terms of understanding your results)? Explain.

7. To transform bacteria with plasmids, technicians first make the bacteria competent (capable of taking upDNA) by placing them in calcium chloride and chilling them. Plasmid is then added to the competentbacteria and the plasmid/bacteria combination is taken through a few more steps to make the bacteriatake up the DNA. In your experiment, should you treat a tube of bacteria that you do not add plasmid toexactly as you do the tube of bacteria that you will transform? Why or why not?

Other things to consider in planning

1. What controls will you include in your experiment?

a. How will you differentiate between bacteria that have taken up the plasmid and those that have not?

b. Are there any controls you can include which would help you determine why the transformation did not work if it does not?

c. What controls could you include to definitively demonstrate that any phenotypic changes you see are adirect result of the introduction of the plasmid into the bacteria?

2. What do you expect to observe, given how you have set up the experiment?

3. Draw any tables or any other method for organizing the data you will collect. Remember that if you collectdata from other groups, you will have to have a way of recording their data as well.



Transformation Laboratory ProcedureAfter discussing your experimental designwith the class, it is time to conduct the actualtransformation to determine the identity ofthe plasmids. Each group will be assigned oneplasmid to work with.

Remember the following:

• One plasmid has a kanamycin-resistancegene.

• Two plasmids have an ampicillin-resistancegene.

• In addition to having an antibiotic-resistance marker, one plasmid also codesfor the gene for green fluorescent protein(GFP), a protein from the bioluminescentjellyfish Aequorea victoria.

Materials

For your group:

2 LB plates

2 kanamycin plates

2 ampicillin plates

2 sterile transformation tubes

container with crushed ice

rack for holding transformation tubes

3 sterile inoculating loops

6 sterile transfer pipets

waste container

3-mL vial LB

3-mL vial CaCl2 (on ice)

To share:

glass beads for spreading

water bath (42°C)

incubator (if used)

starter plate (shared between two groups)

plasmids (will be marked "Plasmid 1," "Plasmid 2," or "Plasmid 3")



Procedure

When performing all of the following steps use sterile technique.

1. Mark one sterile 15-mL tube “+plasmid.” Mark another “–plasmid.” PlasmidDNA will be added only to the +plasmid tube.

2. Use a sterile transfer pipet to add 250 µL of ice-cold calcium chloride to eachtube. See the figure at the right for reference. Note: Pressing the conical areabetween the stem and bulb of the pipet provides better control of the amountof liquid being aspirated.

3. Place both tubes on ice.

4. Use a sterile disposable inoculating loop to transfer isolated colonies of E. colifrom the starter plate to the +plasmid tube. The total area of the coloniespicked should be about half the size of the top of a pencil eraser (the ball ofcells on the loop should be approximately 1–2 mm in diameter.)

a. Do not transfer any agar from the plate along with the cell mass.

b. Immerse the cells on the loop in the calcium chloride solution in the +plasmidtube and vigorously spin the loop in the solution to dislodge the cell mass. Hold the tube up to the light to verify that the cell mass has fallen off the loop.

5. Immediately suspend the cells by repeatedly pipetting in and out with a steriletransfer pipet. It is not necessary to draw the suspension all the way up into the tube; keep the solution in thestem of the pipet. Examine the tube against the light to confirm that no visible clumps of cells remain in thetube or are lost in the bulb of the transfer pipet. The suspension should appear milky white.

6. Return the +plasmid tube to ice.

7. Transfer a mass of cells to the –plasmid tube and resuspend as described in steps 4 and 5 above.

8. Return the –plasmid tube to ice. Both tubes should now be on ice.

9. Use a new sterile disposable inoculating loop to add one loopful of plasmid DNA to the +plasmid tube only.When the DNA solution forms a film across the loop opening, its volume is 10 µL. If you do not see this film,you do not have enough DNAon the loop. Immerse theloopful of plasmid DNAdirectly into the cell suspensionand spin the loop to mix theDNA with the cells.

10. Return the +plasmid tube toice and incubate both tubes onice for 15 minutes.

11. While the tubes areincubating, label your mediaplates as indicated in thediagram and with your labgroup name and date. Thinkabout why you are labeling these plates as you are.


500 µL (0.50 mL)

100 µL (0.10 mL)

250 µL (0.25 mL)

750 µL (0.75 mL)

1,000 µL (1.00 mL)


12. Following the 15-minute incubation on ice, heat shock the cells as follows. Remove both tubes directlyfrom ice and immediately immerse them in the 42°C water bath for 90 seconds. Gently agitate the tubeswhile they are in the water bath. Return both tubes directly to ice for 1 minute or more. The abrupttransfer of the tubes from ice to 42°C and back again is critical.

13. Use a sterile transfer pipet to add 250 µL Luria broth (LB) to each tube. Gently agitate the tubes with yourfinger to mix the LB with the cell suspension. Allow the tubes to sit at room temperature for a 5–15 minuterecovery. During this incubation your instructor may have you add the glass beads for spreading yourbacteria to your plates. See 14a. for instructions on how to do this.

14. Using the procedure below, spread the cells in the +plasmid tube onto the +plasmid plates and the cells inthe –plasmid tube onto the –plasmid plates.

a. Place the plates lid side down. Slightly open the plate on one side and carefully pour 4–6 glass beads onto each plate. Flip the plate over so that the beads are now resting on the agar.

b. Using a sterile transfer pipet add 100 µL of cells from the –plasmid transformation tube to each of the appropriate plates.

c. Using a new sterile transfer pipet add 100 µL of cells from the +plasmid transformation tube to each of the appropriate plates.

d. Spread the bacteria evenly across the plate by using a back-and-forth motion (not swirling round and round) to move the glass beads across the entire surface of each plate. Shake each plate for several minutes.

e. When you finish spreading, let the plates sit for several minutes to allow the agar to absorb the cell suspension.

f. To remove the glass beads, hold each plate vertically over a waste container, slightly open the lower edge, and tap out the beads into the container.

15. Stack your plates together and incubate them as instructed by your instructor.

Laboratory Questions

1. Record your results and conclusions using the organizational method you devised in the Pre-laboratoryInquiry Activity.

2. Did you observe what you expected to? If not, how would you explain your observations?



3. The results from three different experiments are as described in a, b, and c. Something has gone wrongwith each of these experiments. Use the controls to figure out what has gone wrong in each experiment.

a. Results from Experiment 1:

Plate ResultsLB/amp+plasmid plate (no lawn, no colonies)LB/amp–plasmid plate (no lawn, no colonies)LB/kan+plasmid plate (no lawn, no colonies)LB/kan–plasmid plate (no lawn, no colonies)LB+plasmid plate (no lawn, no colonies)LB–plasmid plate (no lawn, no colonies)

Explanation:

b. Results from Experiment 2:

Plate ResultsLB/amp+plasmid plate (lawn)LB/amp–plasmid plate (lawn)LB/kan+plasmid plate (clean plate)LB/kan–plasmid plate (clean plate)LB+plasmid plate (lawn)LB–plasmid plate (lawn)

Explanation:

c. Results from Experiment 3:

Plate ResultsLB/amp+plasmid plate (colonies)LB/amp–plasmid plate (colonies)LB/kan+plasmid plate (clean plate)LB/kan–plasmid plate (clean plate)LB+plasmid plate (lawn)LB–plasmid plate (lawn)

Explanation:



4. Having a way to measure transformation efficiency helps in discussing results or in comparingtransformations that were not done at the same time. Transformation efficiency is expressed as thenumber of transformed colonies (in this case those that are antibiotic-resistant) per microgram of plasmidused in the transformation.

a. Figure out how you would calculate transformation efficiency (i.e., number of colonies/µg of plasmid used). You used 10 µL of plasmid at a concentration of 0.005 µg/µL.

b. Now use the method you devised above to determine the transformation efficiency for the transformation performed by your group.

c. What might be sources of error in calculating this number?

5. You are making ampicillin plates. Before pouring the plates, you add 2 mL of 10 mg/mL to the 400-mL bottle of LB agar that you use to pour the plates. What is the final concentration of the ampicillin in the plates? Express your answer as µg/mL. Show your work.

6. Again, you are making ampicillin plates using LB agar. You are given a vial of ampicillin that is labeled as a1% solution and told that you need to make 40 plates using this solution. Assuming that you will need 25 mL per plate, what volume of LB agar solution should you make to prepare the 40 plates? What volumeof the 1% ampicillin solution do you need to add to this volume of LB agar if the final concentration ofampicillin in the plates should be 50 µg/mL? Hint: Percentage is an expression of weight of solute pervolume of solution.



Big Idea Assessments

1. Give a specific example of how the introduction of a gene into a bacterium can change the phenotype ofthe bacterium. Also explain the specific role of the protein expressed by the gene in changing thebacterium's phenotype.

2. Griffin performed experiments demonstrating that when live, nonpathogenic, S. pneumoniae (whichproduce rough-surfaced colonies) are mixed with killed smooth-surfaced S. pneumoniae (which arepathogenic when alive) and are then injected into mice, the mice become ill. Bacteria isolated from thesesick mice form the smooth colonies characteristic of the pathogenic strain. What happened to the bacteriato make them pathogenic to the mice?

3. Briefly describe how the experiments of Avery, McCarty, and MacLeod, building on the work of Griffith,demonstrated that DNA was the molecule that passed on traits.

4. You have isolated one of the genes for producing one of the blood-clotting proteins needed by somehemophiliacs. Briefly describe how you could create bacteria that would produce this protein.

5. Name the two ways in which bacteria can acquire new genetic material. Both ways are examples of lateral(or horizontal) gene transfer.



6. Assume that you transform bacteria with a plasmid containing an ampicillin-resistance gene. Instead ofdirectly plating the transformed population as you did in this lab, you set up two liquid cultures of them,one that contains ampicillin and one that does not. You will then assay these cultures on plates at twodifferent times: immediately after you set up the cultures, and then again after the bacteria have been inculture for an extended period. The assays will demonstrate the number of ampicillin-resistant vs.ampicillin-sensitive bacteria in each culture at each time. To perform each of the two assays, you prepareserial dilutions of the two cultures and plate them onto LB plates with and without ampicillin (the dilutionis simply to ensure that you will get some plates on which you can distinguish separate colonies).

Describe what you expect to observe in the initial assay and in the second assay. What, if any, differencesmight you expect in terms of the ratios of ampicillin-resistant and nonresistant bacteria?



Appendix

Sterile Technique

Because many microorganisms grow under the same conditions as E. coli, it is important to maintain sterileconditions that minimize the possibility of contamination with foreign bacteria, mold, or fungi. In thisexperiment, the calcium chloride, LB broth, transfer pipets, inoculating loops, culture tubes, and petriplates are sterile. There is no need to flame the plastic ware. Flaming is necessary only for sterilizing thewire inoculating loop when streaking the starter plate(s.)

Good sterile technique involves the following and should be used throughout the experiment.

1. Always open the wrappers of the sterile pipets and loops so that you do not touch the working end. Open apipet wrapper from the bulbous end of the pipet; open a loop wrapper from the pointed end of the pipet.

2. Never allow the unwrapped circular loop, or the narrow end of the pipet to contact any nonsterile object.

3. Do not reuse pipets or transfer loops. To avoid contaminating your work surface, once materials have comeinto contact with E. coli do not place them onto your working surface but in a waste container.

4. Wash your hands thoroughly before and after working with cultures.

5. Wipe your work area with 10% bleach or 70% ethanol before and after working.

6. When transferring things to or from a tube or vial, have the loop or bulb pipet ready in one hand. Pick upthe tube or vial you are transferring to or from and quickly remove the cap. Some people remove and holdthe cap using the pinkie finger of the hand holding the loop or bulb. Others are more comfortableremoving and holding the cap using the thumb and forefinger of the hand holding the vial or tube.Whichever technique you use, do not drop the cap or put it down, and make sure that you hold it with theopen side facing down. Also, make sure you keep your fingers away from the rim of the cap. Replace thecap quickly once you have made your addition to the tube.

7. When putting loops or transfer pipets into tubes or vials while transferring material do not touch the sidesof the tube or vial.

8. When adding things to or collecting bacteria from agar plates, hold the lid over the plate to preventcontaminants from falling onto the surface of the plate. Open the plate the minimum amount needed toperform the manipulation.



Appendix

Data Tables

1. Count the number of colonies on each plate and record your results in the table below. If there is acontinuous, or nearly continuous, lawn of bacterial growth write "lawn." Use the data presented by yourclassmates to fill in the parts of the table for which you have no data. There may be multiple sets of datafor each plasmid. Circle the data from your group's experiment.

2. Use the data in the table above to fill in the table below.


+ plasmid – plasmid + plasmid – plasmid + plasmid – plasmid Color of colonies(where they appear)

Plasmid 1

Plasmid 2

Plasmid 3

LB AMPICILLIN PLATES LB KANAMYCIN PLATES LB PLATES

Does the plasmid belowcontain

an ampicillin-resistancegene?

a kanamycin-resistancegene?

a green fluorescentprotein gene?

Plasmid 1

Plasmid 2

Plasmid 3

Carolina Biological Supply Company

2700 York Road

Burlington, North Carolina 27215

Phone: 800.334.5551

Fax: 800.222.7112

Technical Support: 800.227.1150

www.carolina.com

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*AP and Advanced Placement Program are registered trademarks of the College Board, which was not involved in the production of, and does not endorse, this product.

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