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PIKES PEAK COMMUNITY COLLEGE

COURSE SYLLABUS

Course ID: BIO 224 Course Title: Genetics

Term: Fall 2013

Credit Hours: 4 Contact Hours: 75

Class Times: Lecture

Lab

Faculty Information: Name:

E-Mail Address:

Office Location Campus: Room:

Office Phone #:

Office Hours: TBA

Division Office Contact Phone: Health & Sciences (719) 502-3400

Room F300 – Centennial Campus

Room W209 – Rampart Range Campus

Course Description: Studies the fundamental laws of heredity and their application to living

organisms. Covers the basics of genetics. Focuses on the laws of Mendel, linkage, mutation

concept, molecular genetics, and the Hardy-Weinberg law. This course includes laboratory

experience.

Prerequisites/Co-Requisites: BIO 111 General College Biology I with Lab: SC1

Course Materials:

- Genetics,3rd Edition by Brooker et al

- Packet of Scantron sheets

OPTIONAL Course Materials:

- Basic calculator

- Inexpensive set of colored pencils

STANDARD COMPETENCIES:

I. Demonstrate an understanding of the structure and function of DNA.

II. Demonstrate an understanding of Mendel‟s laws of inheritance and genetic concepts outside of

Mendelian genetics.

III. Demonstrate familiarity with techniques and equipment found in a genetics lab.

IV. Interpret a human karyotype.

V. Demonstrate an understanding of the special genetics of bacteria and viruses.

VI. Demonstrate an understanding of the genetic component of cancer.

VII. Demonstrate an understanding of extranuclear genetics.

VIII. Apply the Hardy-Weinberg Equilibrium to population genetics.

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TOPICAL OUTLINE:

I. DNA Structure and Manipulation.

II. Transmission Genetics.

III. Gene and Chromosomal Structure.

IV. DNA Replication and Recombination.

V. Mutation and DNA Repair.

VI. Human Karyotypes and Chromosome Behavior.

VII. Genetics of Bacteria and Viruses.

VIII. Gene Expression and Regulation.

IX. Genetic Control of Development.

X. The Cell Cycle and Cancer.

XI. Extranuclear Inheritance.

XII. Population Genetics and Evolution

Course Guidelines:

Three hours a week will be devoted to lecture and discussion. Participation is

essential; an extensive amount of material is covered each week.

PPCCConnect (D2L) Access: All students have access to the materials posted on the D2L

course website through the Internet. Each student will receive written instructions to allow

them to access this website either through the college computer lab access or from home.

Any student who is unfamiliar with the Internet should ask for hands-on instruction.

It is especially important to be present for ALL labs. In most cases, lab work will be done

with a lab partner and they may have difficulty completing the work alone. Although

partners work together setting up each lab and interpreting the data, it is expected that

each partner will record their own observations and formulate their own reports.

Plagiarism is the representation of another’s work as your own and will not be tolerated.

Complaints from lab partners about copying of their work are taken seriously.

Plagiarism of all or part of a lab will result in a grade of zero points on the entire

affected assignment.

Academic Standards and Grading: See the current PPCC Catalog

(http://www.ppcc.edu/CatalogSchedule/CurrentCatalog/ and click on the “Academic

Standards” link) for important information regarding academic standards and the grading

system that applies to this course.

Americans With Disabilities Act (ADA)

Any student eligible for and needing academic accommodations because of a disability is

requested to speak with the Office of Accommodative Services and Instructional Support

(OASIS) (502-3333) The following link provides additional information:

www.ppcc.edu/OASIS.

Student Conduct: Review the policies on student conduct in the college catalog (at

http://www.ppcc.edu/CatalogSchedule/CurrentCatalog/ and click on the “Student Conduct”

link).

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Withdrawals: Drop with a refund is possible during the first 15 percent of the term. An

official withdrawal may also be initiated by the student through 80 percent of the term

resulting in a grade of “W.” A “W” grade has no credit and is not computed in the GPA. If

you simply stop attending without officially withdrawing, a grade based on the total points

earned will be assigned to you at the end of the term as per the grading policy listed in the

syllabus. This will usually result in an “F” grade on your grade report and may not be

changed to a “W” once it is issued. Important Note: Withdrawal for any reason after the

official term and will result in the student forfeiting the Colorado College Opportunity Fund

(COF) credit in an amount equal to this course‟s credit hours.

Incomplete: An Incomplete “I” grade will be issued only if the student has completed more

than 75 percent of the course requirements, and has an emergency that cannot be resolved

prior to the end of the semester. An Incomplete “I” is rarely issued and may pose some risk to

your GPA. All remaining work must be satisfactorily completed by the contracted date prior

to the end of the next semester or a grade of “F” will be issued for the course.

Audit: Students may choose to take this course for audit. Normally, the audit option should

be declared at registration; however, students may change their registration from credit to

audit up to the current term “Drop Date” (first 15% of the term) published in the PPCC

schedule. The request to change to audit must be done on a semester registration form and

must receive written recommendation by the Division Dean and be approved by the Vice

President for Educational Services prior to the published “Drop Date.” Once an audit status is

approved, the decision is irreversible. Audit grades do not transfer and are not computed in

the GPA. Courses taken by audit do not count toward enrollment status for financial aid or

veterans‟ educational benefits. Important Note: Audit courses are not eligible for the

Colorado College Opportunity Fund (COF) stipend. Students are responsible for the

additional tuition amount per credit hour audited that would normally be covered by COF.

Lab Safety: It is imperative that students use safe laboratory techniques at all times. Failure

to adhere to the safety requirements in lab will result in the dismissal of the student from the

lab to safeguard the entire class.

Classroom civility: Students must attend regularly and arrive on time. Late arrivals disrupt

the class and interfere with other students‟ ability to concentrate and learn. Please leave cell

phones or pagers at home, or be sure they are turned off. Sleeping or eating during class,

doing homework for other courses, reading assignments for other courses and repeatedly

getting up and leaving the class (unless you are sick, of course) communicates indifference

towards this subject, your classmates and their commitment to this class. Food or drinks are

not permitted in the laboratory.

Cell phones: Must be turned off and put away during class. ABSOLUTELY NO TEXT-

MESSAGING DURING CLASS.

D2L PPCCConnect access: All students have access to the materials posted on the

Blackboard Vista course website through the internet. Each student will receive written

instructions to allow them to access this website either through the college computer labs or

from home. Any student unfamiliar with using the internet should ask for hands on

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instruction. This is a course requirement and any student unable to meet this requirement

must meet with the instructor on the first day of class to discuss alternatives.

Snow Closure Information: Call 719-502-2000 (switchboard) or online at www.ppcc.edu.

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BIO 224 Lecture Schedule: Note: Schedule Subject to Change Week Date Lecture Topic Chapter 1 8/23 Genetics Overview 1 Mendelian Inheritance 2 2 8/30 Reproduction & Chromosome Transmission 3 Extension of Mendelian Inheritance 4 3 9/6 Linkage & Mapping 5 4 9/13 Gene Transfer and Mapping in Bacteria & Phage 6 Non-Mendelian Inheritance 7 EXAM I (Ch 1-7) 5 9/20 Chromosome Structure 8 DNA & RNA Structure 9 6 9/27 Chromosome Organization 10 DNA Replication 11 7 10/4 Gene Transcription 12 RNA Translation 13 EXAM 2 (Ch 8-13) 8 10/11 Gene Regulation in Bacteria 14 9 10/18 Gene Regulation in Eukaryotes 15 10 10/25 Mutation 16 Recombination 17 11 11/1 Recombinant DNA Technology 18 EXAM 3 (Ch 14-18) 12 11/8 Biotechnology 19 13 11/15 Linkage & Genomics 20 14 11/22 Medical Genetics and Cancer 21 Developmental Genetics 22 15 11/29 Population Genetics 24 Evolutionary Genetics 26 EXAM 4 (Ch 19-22, 24 & 26)

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BIO 224 Lab Schedule Week Saturday Lab Topic Experiment 1 8/28 Mono- and Dihybrid Crosses 2A & 2B 2 9/4 Practice Problems - Mendelian Genetics Handout 1 3 9/11 Lab Safety, Sex-Linked Genes, Linkage 3A, 5A & 5B & Mapping, Human Karyotyping 4 9/18 X-inactivation 7A 5 9/25 DNA & RNA Structure 9A & 9B 6 10/2 DNA Replication 11A 7 10/9 mRNA Translation 13A & 13B 8 10/16 Gene Regulation 14A, 15A & 15B 9 10/23 Mutation 16A Start Bacterial Transformation Lab 10 10/30 Recombination 17A Bacterial Transformation Lab (cont.) 11 11/6 Recombinant DNA Technology & Biotechnology 18A & 19A Start Sturgeon DNA Analysis Lab 12 11/13 Linkage & Genomics 20A Sturgeon DNA Analysis Lab (cont.) 13 11/20 Medical Genetics and Cancer 22A Sturgeon DNA Analysis Lab (cont.) 11/27 NO LAB - Thanksgiving Holiday 14 12/4 Hardy-Weinberg Population Genetics IN-LAB handout 15 12/11 Evolutionary Genetics 26A

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BIO 224 The LAC Operon Worksheet Name ______________________

Work in groups to answer the following questions.

1. The operon model for the regulation of enzyme synthesis concerned in lactose utilization

by E. coli includes a regulator gene (I), an operator region (O), a structural gene (Z) for

the enzyme beta-galactosidase, and another structural gene (Y) for beta-galactoside

permease. Beta- galactoside permease transports lactose into the bacterium, where beta-

galactosidase cleaves it into galactose and glucose. Mutations in the lac operon have the

following effects:

Z- and Y- mutant strains are unable to make functional beta-galactosidase and beta-

galactosidase permease

I- and Oc mutant strains synthesize the lac operon gene products constitutively.

The following diagram is a partially diploid strain of E. coli that carries two copies of the lac

operon. On the diagram, fill in the genotype that will result in the constitutive synthesis of

beta-galactosidase and the inducible synthesis of beta-galactoside permease by this partial

diploid.

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2. Chapter 18 of your text describes a blotting method known as Northern blotting, which

can be used to detect RNA transcribed from a particular gene or a particular operon. In

this method, a specific RNA is detected by using a short segment of cloned DNA as a

probe. The DNA probe, which is radioactive, is complementary to the RNA that the

researcher wishes to detect. After the radioactive probe DNA binds to the RNA within a

blot of a gel, the RNA is visualized as a dark (radioactive) band on an X-ray film. For

example, a DNA probe complementary to the mRNA of the lac operon could be used to

specifically detect the lac operon mRNA on a gel blot. As shown here, the method of

Northern blotting can be used to determine the amount of a particular RNA transcribed

under different types of growth conditions. In this Northern blot, bacteria containing a

normal lac operon were grown under different types of conditions, and then the mRNA

was isolated from the cells and subjected to a Northern blot, using a probe that is

complementary to the mRNA of the lac operon.

Based on your understanding of the regulation of the lac operon, explain these results. Which

is more effective at shutting down the lac operon, the binding of the lac repressor or the

removal of the CAP? Explain your answer based on the results shown in the Northern blot.

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3. As described in Question #2 and Chapter 18, the technique of Northern blotting can be

used to detect the transcription of RNA. Draw the results you would expect from a

Northern blot if bacteria were grown in media containing lactose (and no glucose) but had

the following mutations:

Lane 1: Normal strain

Lane 2: Strain with a mutation that inactivates the lac repressor

Lane 3: Strain with a mutation that prevents allolactose from binding to the lac repressor

Lane 4: Strain with a mutation that inactivates CAP

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4. Discuss the advantages and disadvantages of genetic regulation at the different levels

described in the figure below.

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Population Genetics Lab

Hardy-Weinberg Equilibrium

Population Genetics

A. Create a Punnett Square for a mating between an individual with genotype AA and an

individual with genotype aa. Show the genotypes of the offspring in their expected

proportions.

B. Define evolution with respect to genotypes and alleles.

C. List the five mechanisms of evolution.

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In the following exercises, you will find out what happens to the allele and genotype frequencies

of a population when each of the mechanisms of evolution acts upon a population.

Use the wooden disks marked with “A” and “a” to represent alleles in each population. You

should have 40 A alleles and 40 a alleles total.

I. Mechanism 1

A. Suppose there exists a population of 10 individuals, all with the genotype AA. What are the

genotype and allele frequencies in this population?

B. Suppose two individuals have changes in the DNA of their germ lines, changing an A allele to

an a allele. Every individual in the population then has two offspring, which are clones of the

parent. What are the genotype and allele frequencies in the second generation?

C. Was there a change in genotype or allele frequency in this population from one generation to

the next? Did evolution occur?

D. What was the mechanism of evolution acting on this population?

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II. Mechanism 2

A. Suppose there exists a population of 10 individuals: 5 AA and 5 aa. What are the genotype and allele frequencies in this population?

B. Suppose 2 aa individuals move out of the population. Each remaining individual has two offspring, which are clones of the parent. What are the genotype and allele frequencies in the second generation?

C. Did the allele or genotype frequencies change in this population from one generation to the next? Did evolution occur?

D. What was the mechanism of evolution acting on this population?

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III. Mechanism 3

A. Suppose there exists a population of nine individuals: 3AA, 3Aa and 3aa. In the first

generation, all of these individuals mate with each other regardless of genotype or phenotype.

Fill in the missing values in the table below, showing the number of offspring of each

genotype produced by each mating pair. Assume each pair produces four offspring.

Mating Pairs Expected Offspring Genotypes

AA Aa aa

AA x AA 4

AA x Aa

AA x aa 4

Aa x AA

Aa x Aa 1 2 1

Aa x aa 2 2

aa x AA

aa x Aa

aa x aa 4

Total #

Total %

B. In the second generation, individuals mate only with others who are genotypically different.

Fill in the table below for the expected offspring genotypes for each mating pair that produces

four offspring.

Mating Pairs Expected Offspring Genotypes

AA Aa aa

AA x aa

AA x Aa

Aa x AA

Aa x aa

aa x AA

aa x Aa

Total #

Total %

C. Did the frequency of offspring genotypes change from one generation to the next?

D. Which genotypes increased in frequency, and which decreased? Explain why this pattern

appeared.

E. Which mechanism of evolution was acting on this population?

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IV. Mechanism 4

A. Suppose there exists a population of 40 individuals: 10 AA, 20 Aa, and 10 aa. What are the

genotype and allele frequencies in this population?

B. Suppose there is one offspring to replace each individual in the next generation. Randomly

create 40 new offspring from the alleles of the parents. Do this by putting all alleles in a bag,

then draw two alleles at a time, and record the genotype. Put the alleles back in the bag, and

repeat the process until you have recorded 40 offspring. What are the genotype and allele

frequencies in the second generation?

C. Did the allele or genotype frequencies change in this population from one generation to the

next? If so, why?

D.What was the mechanism of evolution that acted on this population?

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V. Mechanism 5

A. Suppose there exists a population of 40 individuals: 10 AA, 20 Aa, and 10 aa. What are the

genotype and allele frequencies in this population?

B. Suppose the genotype aa gives rise to a lethal disease that causes the death of the individual

before it can reproduce. Which genotypes will donate alleles to the next generation?

C. If the surviving genotypes mate randomly, what will be the possible genotype matings and

their offspring?

D. Has the a allele been eliminated from the population? Why could the a allele continue to be

present in the population without being completely eliminated?

E. Which mechanism of evolution has acted on this population?

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Genetic Analysis of Sturgeon Caviar

Adapted from: Kathleen A. Nolan, Nancy Rosenbaum, Claire Leonard, Anthony Catalano, Phaedra Doukakis, Vadim Birstein, and Rob DeSalle. 2004. Introducing students to conservation genetics using sturgeon caviar and other fish eggs. Pages 85-97, in Tested studies for laboratory teaching, Vol. 25 (M.A. O’Donnell, Editor). Proceedings of the 25th Workshop/Conference of the Association for Biology Laboratory Education (ABLE), 414 pages.

BIO 224

Genetics

Fall 2013

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Contents General Notes…………………………………………………………………………… 2

Lab 1: Overview of Project and DNA Isolation………………………………………… 3

Lab 2: Polymerase Chain Reaction……………………………………………………… 10

Lab 3: Gel Electrophoresis………………………………………………………………. 17

Lab 4: Interpreting the Gels.……………………………………………………………..

Lab 5: Bioinformatics……………………………………………………………………..

General Notes The steps should be followed exactly for each procedure in this manual. You must read

through all of the steps before beginning any of the procedures. If you have questions about a

procedure, you should ask before you begin the procedure.

You must follow all safety precautions described in the procedures. Failure to do so will

result in dismissal from the lab.

Each group will present their findings from these exercises in a professional oral presentation

for a grade. Guidelines for the presentation are posted on the course webpage on D2L.

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Lab 1: Overview of Project and DNA Isolation

Objectives 1. Describe the general purpose of the project “Genetic Analysis of Sturgeon Caviar”.

2. State the specific goal of the project.

3. Isolate DNA from fish eggs.

4. Explain, in general terms, what it means to isolate DNA.

Before Lab 1. Read the lab exercise.

2. Complete the pre-lab assignment found on page 8 (due before lab on Tuesday.)

Introduction Fish eggs, or roe, are a delicacy in many cuisines of the world. The unqualified term “caviar”

refers specifically to eggs from sturgeon fish (family Acipenseridae) (Food and Drug

Administration, 2009). Caviar is a luxury item that can sell for thousands of dollars per

pound in the open market. There are many culinary substitutes (such as eggs from paddlefish,

salmon, or whitefish) that have similar textures and flavors, but do not carry the exorbitant

cost of eggs from sturgeon. Occasionally, eggs from other fish are (intentionally or not)

marketed as eggs from sturgeon.

Historically, the extraction of eggs from sturgeon resulted in the death of the fish. In recent

years, some fisheries have found ways of obtaining the eggs without killing the female fish

(Prince, 2009). This new process is still not widespread in use. The death of sturgeon fish is

of particular concern, because of the decline in population numbers of these fish. Cullen

(1999) describes the decline of several species of caviar-producing sturgeon for commercial

purposes in the Caspian Sea. American caviar-producing species are also in decline in areas

such as the Hudson River. There is currently a moratorium on sturgeon fishing in the Hudson

River for the next forty years. All twenty-five species of sturgeons and paddlefishes (order

Acipenseriformes) are threatened by over-fishing and habitat degradation (Birstein et al.,

1998).

Most species of sturgeon do not reproduce until they are approximately fifteen years old,

which means that sturgeons are very slow-growing fish. Unfortunately, they are often

harvested before they are even at this reproductive age, which contributes even more to their

decline in abundance. The three species that produce the caviar that is most often found in

U.S. delis (and now over the internet) are the beluga (Huso huso), the sevruga (Acipenser

stellatus) and the ossetra (Acipenser gueldenstaedti).

As caviar commands a high price in the marketplace, these fish are sometimes illegally

caught.

All sturgeon species were placed on the Convention for Trade in Endangered Species

(CITES) list in 1998. Sometimes a species of sturgeon that produces “cheaper” caviar is

substituted for a species that produces more expensive caviar. Worse, sometimes caviar from

the more “commonly available” sturgeon are replaced by even rarer species.

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DeSalle et al. (1996) reported that of twenty-three lots of caviar purchased in Manhattan delis

and two lots from Russia, five were incorrectly labeled, according to species-specific primers

that have been developed. This was ascertained from DNA that was isolated from a single

egg. Quick methods of species identification of sturgeon as well as other organisms may

reveal other misidentifications as well. Ultimately this work may put pressure on sellers and

consumers alike to conserve our natural resources.

Overview The goal of this series of laboratory exercises is to use techniques in genetics and

biotechnology to determine the accuracy of labeling of commercially available caviar.

Specifically, students will determine whether or not containers labeled with “sevruga caviar”

contain eggs from the sevruga sturgeon (A. stellatus). The working hypothesis is that the eggs

in the container are correctly labeled.

Students will isolate DNA from caviar, then amplify and visualize a fragment from the

sturgeon cytochrome b mitochondrial DNA gene, using polymerase chain reaction (PCR) and

gel electrophoresis. Advanced techniques may include isolating the DNA for sequencing, and

then using bioinformatics to identify the exact species of fish from which the eggs were

obtained. The general purpose of each lab is described below.

Lab 1: Overview of Project and DNA Isolation

You will gain a general understanding of the techniques used during the subsequent labs.

Then, the DNA from eggs labeled “sevruga caviar” will be separated out from the other

cellular components. This DNA will be used in the subsequent labs for analysis.

Lab 2: PCR

When DNA is isolated from a source, there is usually a miniscule amount that is obtained

from the process. A much greater amount is needed in order to analyze the DNA using

biotechnology. One function of PCR is to amplify (make many identical copies of) DNA. A

second function of PCR is to further isolate fragments of interest of DNA. You will try to

isolate a fragment of DNA from the mitochondrial cytochrome b gene of sevruga sturgeon.

This particular fragment is unique to sevruga sturgeon, and is not found in other kinds of

sturgeon. If you are able to isolate and amplify this fragment, then the DNA from your egg

comes from A. stellatus. If you are not able to isolate this particular fragment, then the DNA

likely did not come from A. stellatus.

Lab 3: Gel Electrophoresis

The DNA amplified by PCR is not visible to the naked eye without further manipulation. Gel

electrophoresis allows DNA fragments to be separated by size. The fragments of DNA can

then be stained and seen with the naked eye.

Lab 4: Bioinformatics

Your instructor has isolated DNA…

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Isolating DNA From Caviar Eggs

Background The technique of DNA isolation separates out the DNA from all of the other cellular

components. Recall that DNA is one type of biological macromolecule, made of nucleotide

monomers. DNA must be separated from all of the other macromolecules in a cell, such as

lipids, carbohydrates, and proteins. A proprietary mix of chemicals, called DNAzol, will be

used to precipitate the DNA from the eggs.

Only nuclease-free water will be used in the procedure. Nucleases are enzymes that break

down RNA and DNA. Since the DNA must be preserved in the experiment, there must be no

errant enzymes lurking in the water that can interfere with the procedure.

Lab Safety 1. Wear closed-toe shoes, and tie back long hair.

2. Clean all surfaces before and after the procedure.

3. Wear gloves during the entire procedure. Oils and products from your hands can interfere

with the process, and reagents can be toxic.

4. Discard pipette tips, tubes, and liquids only in the disposal beakers provided.

Materials Per Group

Quantity Item

2 1.5 mL microfuge tubes

1 microfuge tube rack

250 μL nuclease-free sterile water

1 sturgeon egg from commercially available sevruga caviar

1 ea. 1000 μL, 100 μL, 20 μL pipettes

1 box ea. sterile pipette tips (500 μL, 20 μL and 1 μL)

1 mL DNAzol (reagent for DNA isolation)

500 μL 100% ethanol

1 supernatant/pipette tip disposal beaker

1 mL 70% ethanol

1 permanent marker, fine

For Class

Quantity Item

1 microcentrifuge (up to 14K rpm)

1 box ea. latex-free gloves, sm., med., and large

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Procedure 1. Work in groups. You may divide up the activities within your group, as long as each

student masters all of the skills and concepts.

2. The DNA that you isolate today in lab will be used for the rest of the labs, so it is very

important that you go slowly, be careful and be thorough. Ask questions if you are not

sure about something!

3. Use a new, sterile pipette tip for every transfer of a liquid.

4. Obtain two 1.5 mL sterile microfuge tubes. With a permanent marker, label the tops

and the sides of the tubes with your group‟s identifier (initials, symbol, etc.).

5. Add 50 μL of nuclease-free sterile water to one of the tubes.

6. Add one sturgeon egg to the tube with the water, using a yellow 20 μL pipette tip.

Smash open the egg against the side of the tube. You should see white material oozing

from the egg.

7. Add 1 ml of DNAzol to the tube with the egg, and close the tube securely.

8. Mix the contents by gently inverting the tube five times.

9. Place your group‟s tube in a centrifuge with tubes from other groups. The tubes in the

rotor must be balanced. (If there are an even number of tubes, place the tubes so that

they equally spaced, and any tube is directly opposite another. If there are an odd

number of tubes, fill one extra tube with 50 μL of water, and then space accordingly.)

Put the internal lid on, and close the external lid.

10. Centrifuge at high speed in a microfuge (14,000 RPM) for 10 minutes. This step

removes insoluble tissue fragments, RNA, and excess polysaccharides from the

lysate/homogenate. The DNA will remain in the liquid, while other components will

form a pellet on the bottom of the tube.

11. Transfer all of the supernatant (the liquid) to your other microfuge tube, using a 100 μL

pipette. Be careful not to touch the pellet at the bottom during this process. Discard the

tube with the pellet.

12. Add 500 μL of 100% ethanol to the tube of supernatant and mix well by inverting the

tube 5 to 8 times. Make sure that the DNAzol and the ethanol mix well to form a

homogeneous solution. The solution will be cloudy. Rest at room temperature for 3

minutes.

13. Centrifuge at high speed (14,000 RPM) for 2 minutes. This will precipitate the DNA

from the solution into a pellet at the bottom.

14. Discard all of the supernatant (the liquid), using a 1,000 μL pipette. Be careful not to

touch the pellet at the bottom during this process.

15. Gently add 1 mL of 70% ethanol or isopropanol to the tube with the pellet, to rinse the

precipitate.

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16. Centrifuge for 2 minutes at 14,000 RPM. The microfuge must be balanced. Be sure to

put on the internal lid before closing the centrifuge.

17. Discard all of the supernatant. Be careful not to touch the pellet at the bottom during

this process.

18. Repeat steps 15 through 17.

19. Allow the pellet to air dry until there are no drops of alcohol left. (You will most likely

NOT see a pellet!) *DNAzol Protocol says 15 secs

20. Re-suspend each pellet in 200 μL of DNase-free sterile water. *DNAzol Protocol says

8mM NaOH fresh

21. Store your DNA in a tightly-closed tube in the freezer until next week.

22. Complete the Post-Lab Activity

Works Cited Birstein, V., Doukakis, P., Sorkin, B., and R. DeSalle. 1998. Population aggregation analysis

of three caviar-producing species of sturgeons and implications for the species identification

of black caviar. Conservation Biology 12:766-776.

Cullen, Robert. 1999. The rise and fall of the Caspian Sea. National Geographic 195:2-35.

DeSalle, R. and V. Birstein. 1996. PCR identification of black caviar. Nature 381: 197-8

Food and Drug Administration, 2009. CPG Sec. 540.150 Caviar, Use of Term – Labeling.

(http://www.fda.gov/ICECI/ComplianceManuals/CompliancePolicyGuidanceManual/ucm

074490.htm)

Prince, R. 2009. “Caviar: Golden Eggs”. The Telegraph.

(http://www.telegraph.co.uk/foodanddrink/recipes/4569441/Caviar-Golden-eggs.html)

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Pre-Lab Exercise: Project Overview and DNA Isolation

Name: _______________________________

1. What are the monomers of DNA?

2. What are the goals of the sturgeon caviar project?

3. You will use a centrifuge multiple times in this lab session. What are two particular steps

that you must remember to do before starting the centrifuge?

4. Why is it important to obtain a new pipette tip with every transfer of a liquid?

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Post-Lab Exercise: Project Overview and DNA Isolation

Name: _____________________________

1. The DNA purification process required that the DNA be separated from other cellular

components. List three components of an animal cell that would need to be separated

from the DNA during this process.

2. RNA and DNA are both nucleic acids. The DNAzol used during the isolation process

contained enzymes that were selectively able to break down the RNA, without breaking

down the DNA. Describe the ways in which DNA and RNA are chemically distinct.

3. In the pre-lab exercise, you stated the goals of the project. In your own words, summarize

what each lab in the project will accomplish. (You should use only one or two sentences

to summarize each lab.)

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Lab 2: Polymerase Chain Reaction (PCR)

Objectives 1. Explain the goal of a PCR.

2. Set up a PCR.

3. Explain the role of each component of a PCR.

4. Explain the role of different temperatures in a PCR.

5. Explain the role of PCR in the sevruga caviar project.

6. Describe and explain the positive and the negative controls used in the experiment.

Before Lab 1. Watch the following videos on PCR:

http://www.youtube.com/watch?v=2KoLnIwoZKU

http://www.youtube.com/watch?v=HMC7c2T8fVk&feature=fvwrel

2. Read the PCR lab exercise.

3. Complete the pre-lab assignment, found on page 15 (due before lab on Tuesday.)

Lab Safety 1. Clean all surfaces before and after handling chemicals; do not put anything in your mouth.

2. Wear closed-toed shoes and tie back long hair.

3. Wear gloves during the procedure.

Introduction In the previous lab, you isolated and purified DNA from a single egg labeled as sevruga

caviar. In this lab session, you will attempt to amplify a specific fragment of the DNA using a

polymerase chain reaction (PCR). The following paragraphs explain the concept of PCR and

how you will use it to determine if the DNA from the egg comes from sevruga sturgeon.

PCR is a technique used to produce large amounts of a specific DNA fragment. A low

concentration of target DNA is incubated with two primers (short stretches of single-stranded

DNA) that are complementary to the sequences that flank each end of the target DNA. The

essential components of the reaction are the enzyme DNA polymerase (which copies DNA),

dNTPs (the nucleotides that are connected together by DNA polymerase to synthesize DNA),

a DNA template that includes the DNA segment you want to amplify, and single-stranded

DNA primers (which define the ends of the DNA that will be amplified), and the cofactor

MgCl2.

Suppose you have isolated some ds DNA from a cell. Part of that DNA is shown below. You

wish only to amplify the segment of ds DNA that is underlined.

3‟ …ATC GGG ACC CAC TAT CGT TTT AAA CGT ATC CGT GAC… 5‟

5‟ …TAG CCC TGG GTG ATA GCA AAA TTT GCA TAG GCA CTG… 3‟

27

All of the components listed above (DNA pol, dNTPs, primers, buffers) plus the DNA

containing the fragment to be amplified are put into a small test tube. The test tube is put into

a PCR machine that is programmed to change the temperature of the tubes for precise

amounts of time. The machine cycles through the temperatures multiple times, and at the end,

many copies of the fragments of DNA are produced. How does this happen?

How PCR Works

1. First, the double-stranded DNA must be denatured. This is accomplished by heating the

DNA to a high temperature (> 90OC). The heat breaks the hydrogen bonds between the

bases of the two strands.

2. In a living cell, the enzyme primase creates an RNA primer to begin replication. Since

PCR is done in a test tube, the researcher is in control of what goes into the tube. In PCR,

the researcher puts into the tube two, engineered DNA primers, which are complementary

to both ends of the fragment of interest. The primers are as shown, in bold, below.

3‟ …ATC GGG ACC CAC TAT CGT TTT AAA CGT ATC CGT GAC… 5‟

primer 5’ CCC TGG 3’

3’ ATC CGT 5’ primer

5‟ …TAG CCC TGG GTG ATA GCA AAA TTT GCA TAG GCA CTG… 3‟

The tubes are cooled sufficiently (45- 55OC) to allow the primers to anneal (hydrogen

bond) to the separated DNA. The process works, even if only some of the primers attach

to the DNA before the complementary strands reattach.

3. The tubes are then heated to a moderate temperature (72OC), at which Taq polymerase

works best. Taq polymerase is a type of DNA polymerase isolated from the bacterium

Thermus aquaticus, which lives at very high temperatures. The Taq polymerase is so

stable that it can undergo repeated heating and cooling without a loss of function. At

72OC, Taq polymerase adds the nucleotides to each 3‟ end of a primer sequence,

complementary to the original strand.

By the end of this first cycle, the following strands have been created (the target sequence

is in black type, and extra nucleotides are in blue type):

3‟ …ATC GGG ACC CAC TAT CGT TTT AAA CGT ATC CGT GAC… 5‟

5‟ CCC TGG GTG ATA GCA AAA TTT GCA TAG GCA CTG…..3‟

3‟….ATC GGG ACC CAC TAT CGT TTT AAA CGT ATC CGT 5‟

5‟ …TAG CCC TGG GTG ATA GCA AAA TTT GCA TAG GCA CTG… 3‟

4. The entire cycle is repeated (multiple times). Eventually, DNA containing only the

fragment of interest is amplified more than the DNA containing extra nucleotides on the

end (shown in blue). The second cycle of amplification yields the following fragments:

3‟ …ATC GGG ACC CAC TAT CGT TTT AAA CGT ATC CGT GAC… 5‟

28

5‟ CCC TGG GTG ATA GCA AAA TTT GCA TAG GCA CTG…..3‟

5‟ CCC TGG GTG ATA GCA AAA TTT GCA TAG GCA CTG…..3‟

3‟ GGG ACC CAC TAT CGT TTT AAA CGT ATC CGT 5‟

3‟… ATC GGG ACC CAC TAT CGT TTT AAA CGT ATC 5‟

5‟ CCC TGG GTG ATA GCA AAA TTT GCA TAG 3‟

3‟….ATC GGG ACC CAC TAT CGT TTT AAA CGT ATC CGT 5‟

5‟ …TAG CCC TGG GTG ATA GCA AAA TTT GCA TAG GCA CTG… 3‟

Note that by the end of the second cycle, there are two single strands that contain only the

fragment of interest. The number of strands containing only the fragment of interest

increases with each cycle. If the cycle is repeated 32 more times, then one original copy

of DNA is replicated 232

times. That creates billions of copies of the DNA fragment of

interest.

Project Goal

There are two requirements for PCR to work: first, the primer sequences for the fragment of

interest must be known; second, the fragment of interest must be present in the DNA. If

either of these requirements is not met, then the fragment of interest will not be amplified.

In this exercise, we will use the following primers:

Primer 1 (S2A): 5‟ CCT CCA ATT CAT GTG AGT ACT 3‟

Primer 2 (S2): 5‟ GGA GTC CTA GCC CTC CTG 3‟

When S2 is used with S2A, only a cytochrome b gene fragment (approximately 150 bp)

specific to sevruga sturgeon (A. stellatus) should be amplified. In other words, if the DNA

that you isolated last week is from A. stellatus, then the PCR will amplify the fragment of

interest. If the DNA is from another type of fish, the PCR will not amplify this particular

fragment. Next week, you will do gel electrophoresis in order to see the fragments that were

amplified.

Materials Per Group

Quantity Item

2 1.5 mL microfuge tubes

1 ea. 100 μL, 1 μL pipettors

1 box ea. sterile pipette tips (100 μL, 1 μL)

4 0.2 mL PCR tubes

88 μL PCR Master Mix (Taq PCR Master Mix Kit from Qiagen)

1 μL ea. (S2 and S2A, reconstituted)

200 μL RNase and DNase-free water (for diluting DNA)

1 small tub of ice

29

For Class

Quantity Item

1 thermocycler (PCR machine)

1 microcentrifuge (up to 14K rpm)

4 mL RNase free water (for reconstituting dried primers)

1 4OC freezer (for storing PCR products)

1 box ea. latex-free gloves, sm., med., and large

Procedure 1. Wear gloves, and keep all solutions on ice unless otherwise directed.

2. Use a new pipette tip for each transfer.

3. Since you are not sure of the quantity of DNA that you have isolated, and the PCR reaction

is DNA concentration-dependent, it will be necessary for you to do a dilution series of

your DNA. (It is possible to quantify the amount of DNA directly using specialized

equipment. However, for this exercise, you will simply create a range of DNA

concentrations, using dilutions).

4. Set up two 1.5 mL microfuge tubes for your DNA and label them 1:10, 1:100 on the top.

Also label them with your initials and the date on the sides.

5. Place one μL of your DNA in each tube. Add the appropriate amount of nuclease-free

water to make the dilution. To make a 1:10 dilution, place one part DNA (1 μL) plus nine

parts water (9 μL) into the tube labeled 1:10, and invert gently three times. To make a

1:100 dilution, place one part DNA (1 μL) plus 99 parts water (99 μL) into the tube

labeled 1:100, and invert gently three times.

6. Obtain four 0.2 μL PCR tubes, and label the tops and sides A, B, C, and D. Tubes A and B

will contain the dilutions of your DNA. Tube C will be a positive control, and it will

contain the primers and DNA that is known to be from A. stellatus. Tube D will be a

negative control, and it will contain the primers and no DNA.

Add the substances to each tube, as directed in the following chart:

Amount of Substance to Add to Each Tube

Substance Tube A Tube B Tube C Tube D

DNA 1 μL 1 μL 1 μL none

Taq Master

Mix

22 μL 22 μL 22 μL 22 μL

Primer 1 (S2) 1 μL 1 μL 1 μL 1 μL

Primer 2 (S2A) 1 μL 1 μL 1 μL 1 μL

Nuclease-free

Water

None None None 1 μL

30

The Taq Master Mix has been prepared to contain the following ratios of reactants:22 μL

PCR supermix, containing water, Taq polymerase, PCR buffer supplemented with 15 mM

magnesium chloride, dNTPs (10 mM each).

5. Spin tubes briefly at 14,000 RPM in the microfuge for five seconds.

6. When all groups are ready to run the thermocycler (PCR machine), carry the ice bucket

with the tubes to the thermocycler. Put your tubes into the thermocycler and write down

their exact positions on the diagram provided by your instructor. The labels tend to come

off during PCR, so you need to make sure your tubes are labeled on the top and side, and

that you record the tubes‟ positions in the PCR machine.

Your instructor has programmed the thermocycler to run 35 cycles of the following:

94OC 1 min.

55OC 1 min.

72OC 1 min

7. Your instructor will remove the samples when they are finished, and store them at 4OC

until the following week.

Pre-Lab Exercise: PCR

Name: ______________________________

1. Suppose you wish to amplify the fragment of interest (underlined) on the DNA given

below, using PCR. What would be the two primers you would need to do this, if each

primer were six bp long? Be sure to write each primer in the 5‟ to 3‟ direction.

5‟ GGA CTA CTA TTT GCT TAG AGC CCG TAT 3‟

3‟ CCT GAT GAT AAA CGA ATC TCG GGC ATA 5‟

Primer 1: ________________________________________

Primer 2: ________________________________________

2. Why is it important to use Taq polymerase in the automated process of PCR?

31

3. What is the goal of using PCR in this project?

Post-Lab Exercise: PCR

Name: ______________________________

1. Show three complete PCR cycles of the following DNA segment. The fragment of interest

is underlined.

5‟ CAG TTG CCA ACG 3‟

3‟ GTC AAC GGT TGC 5‟

2. Approximately how many copies of the fragment of interest will be present after 34 cycles

of PCR, starting from one double-strand of DNA? Explain how you determined this.

32

Lab 3: Gel Electrophoresis

Objectives 1. Describe the purpose of gel electrophoresis.

2. Explain why DNA migrates to the positive cathode.

3. Describe the role of a “ladder,” or molecular weight standard.

4. Conduct gel electrophoresis on products from a PCR.

5. Interpret the results of a gel with respect to the sevruga caviar project.

Before Lab 1. Watch the following videos on gel electrophoresis:

http://www.youtube.com/watch?v=wXiiTW3pflM

http://www.youtube.com/watch?v=9f2VSyVhsGI&feature=fvwp&NR=1 (up to “Visualizing the Nucleic Acids”)

2. Read the Gel Electrophoresis Lab handout.

3. Complete the pre-lab assignment found on page 21 (due before lab on Tuesday).

Lab Safety 1. Clean all surfaces before and after handling chemicals; do not put anything in your mouth.

2. Wear closed-toed shoes and tie back long hair.

3. Wear gloves during the procedure.

4. Handle the heated agar with protective pads.

Introduction In the previous lab, you used PCR to attempt to amplify a fragment of the mitochondrial

cytochrome b gene from sevruga sturgeon. In this lab, you will use gel electrophoresis to see

the fragments produced by the PCR and determine their size. If you are able to see a 150 bp

fragment in your gel, then this supports the hypothesis that your DNA came from A. stellatus.

If you are not able to see a 150 bp fragment in your gel, then this will not support the

hypothesis that the DNA came from A. stellatus.

As you saw in the pre-lab videos, gel electrophoresis separates molecules out by their size, or

molecular weights. This is done by moving the molecules through an agarose gel submerged

in a buffer solution with an electrical current running through it. Since DNA is overall

negatively charged, fragments of DNA move toward a positive charge. Since the gel is

porous, the fragments of DNA can move through it. Smaller fragments will move more

quickly through the gel, and travel farther than larger fragments.

The movement of the microscopic DNA is monitored by the dye bromophenol blue. The dye

does not interact with the DNA, but migrates independently toward the positive pole. When

the dye has moved sufficiently toward the positive pole, the procedure can be stopped, and the

DNA can then be stained for visualization.

33

The DNA can be stained using many different methods. The most common method uses

ethidium bromide, a chemical that is a mutagen and carcinogen. For that reason, we will not

be using ethidium bromide in this procedure. A sufficiently effective method for this project

is to stain the DNA with the dye methylene blue, which is less toxic and non-carcinogenic.

The methylene blue interacts directly with the DNA, so wherever the blue dye is found in the

gel, the DNA is found.

Finally, you will determine the sizes of the fragments by comparing them to a DNA “ladder,”

or molecular weight standard. A ladder contains fragments of DNA of known sizes, to which

the unknown samples can be compared.

Materials Per Group

Quantity Item

1 electrophoresis box with 8-12 comb, tray, and power supply

0.75 g agarose

50 ml 1X TBE buffer (adjusted to pH 8)

1 125 mL Erlenmeyer flask

1 ea. 10 μL, 1 μL pipettors

1 box ea. sterile pipette tips

50 μL loading dye (6X bromophenol blue)

10 μL “ladder” (100 bp to 1,000 bp)

1 plastic Tupperware tub with lid (to hold gel)

250 mL 0.002% methylene blue

1 small bucket of ice

1 permanent marker, fine

For Class

Quantity Item

2 hot pads/ heat gloves

1 microwave

1 roll masking tape

1 L 1X TBE buffer (for covering gels)

1 box parafilm

1 microcentrifuge

1 box ea. latex-free gloves, sm., med., and large

1 roll masking tape

distilled water (for destaining gels)

Procedure Making the Gel

1. Wear gloves throughout the procedure, and use heat pads when necessary.

2. Place 0.75 g of agarose into a 125 ml Erlenmeyer flask. Add 50 ml of 1X TBE buffer. If

you use water at this point, your gel will be ruined.

34

3. Swirl the agarose in the liquid and let it absorb the buffer for five minutes.

4. Put a loosely crumpled paper towel in the mouth of the flask to serve as a porous “lid.”

5. Heat to boiling on a hot plate. Keep an eye on your flask, so that it does not spill over.

6. Make sure that all the agarose is dissolved before removing the flask from the hotplate.

The solution should be entirely clear. Use heat pads to handle the flask.

7. Let the gel cool at room temperature until hot but not burning to the touch.

8. Pour the gel according to the directions shown in the video. Use tape to seal the ends.

Make sure your tray is on a level surface. Place an 8-12 well comb into the tray. Pour the

gel quickly, and push any bubbles to the side with a yellow micropipette tip.

9. The gel should be hardened after about 20 minutes; it will turn opaque and you may see

wavy lines running through it.

10. Pull out the comb by wiggling it gently as you pull – this takes a little practice. It helps to

dribble some buffer on top of the gel by the comb. Remove the tape from the ends of the

tray.

Running the Gel

1. Gently place the gel (still in the tray) into an electrophoresis chamber. Be sure that the end

of the gel with the wells is toward the black (negative) anode of the chamber.

2. Cover the gel with 1X TBE buffer. Fill the reservoirs first, then continue to fill until the gel

is covered with about 2 mm of buffer. The less buffer you use, the less resistance to the

current there will be. (If you increase the amount of buffer, you will notice that by

switching to the “current” or “ampere” setting, the number of amperes increases, which is

undesirable because this could create too much heat and cause your gel to melt.)

3. Prepare to load the gel. Take a piece of parafilm around 10-cm square and place it down

flat on the lab bench. Pipet 2 μL of bromophenol blue (loading dye) as dots spaced apart

on the parafilm – one for each sample that you will load. You will have five samples

(one ladder and four PCR products.)

4. Obtain your PCR samples from the previous lab. Spin tubes briefly at top speed in the

microfuge for 10 seconds to collect your samples in the bottoms of the tubes. Put the samples

back on ice.

5. Make a key of the order of your samples that you will be loading into your gel below.

Recall that samples in the outermost lanes do not run as evenly as those in the center.

Avoid using the first or last lanes.

35

Lane # Lane # Lane # Lane # Lane #

Sample

6. To the first dot of loading dye, add 10 μL of the ladder, and mix the sample by pipetting up

and down once. Your pipette should be loaded with your sample.

7. If there is a bubble in the tip of the pipette, very gently push the plunger until the bubble

has been expelled.

8. Steady the pipet over the well using two hands with your elbows on the bench.

9. Place the pipette tip into the chosen well, bing careful not to punch the pipet tip through

the bottom or sides of the well. You only need to place the tip into the top third of the

well.

10. Gently depress the pipette plunger to expel the sample into the appropriate well. Press

only to the first stop of the plunger. The sample may be dislodged from the well if the

plunger is pressed down fully. If the tip is centered over the well, the solution will sink to

the bottom of the well. Leave the plunger depressed as you withdraw the pipette tip; if

you let the plunger come up, you will withdraw the sample.

11. Carefully add your remaining samples to the wells, one at a time. Use a new pipette tip

for each sample. Keep your PCR samples on ice until you are ready to load them.

12. When you have finished loading the samples, and noting which sample went into which

lane, close the top of the electrophoresis chamber, and connect the electrical leads red-red

and black-black. Negative DNA „runs red‟ towards the red + pole. Turn on the power

supply and set the voltage at 100 volts.

13. As the gel runs, you will notice that the loading dye separates out into a dark blue and a

light blue band. The dark blue band travels faster and co-migrates with a DNA fragment

of a little more than 150 bp. The light blue band co-migrates with a DNA fragment of a

larger sized fragment. Let the gel run for 30 to 45 min (this time may vary), or until the

dark blue band has traveled three-quarters the length of the gel. You should keep an eye

on your gel; if the dark blue band runs off of the gel, then you will lose your fragment.

14. Turn off the power supply, remove the gel tray, and carefully slide into a staining chamber

labeled with your initials and the date.

15. Your instructor will put your gel into a staining tray that contains the dye methylene blue.

The staining process will occur overnight. Your instructor will remove the gels and take

digital photos of them with the bands visible. These photos will be posted on the class

website.

36

Pre-Lab Exercise: Gel Electrophoresis

Name: _____________________________

1. What is the general purpose of gel electrophoresis?

2. The gel is submerged in a solution through which an electrical current runs. In which

direction does the DNA move (toward negative or positive anode), and why?

3. What size of fragment (large or small) of DNA moves the farthest through the agarose, and

why?

4. When each of the following errors is made during gel electrophoresis, state the result to the

gel or sample, and explain why.

a. the agarose is not completely dissolved:

b. the sample is loaded into the well and the plunger is pushed to the second stop:

c. the samples are loaded into the wells at the edge (the first or last lanes):

37

Post-Lab Exercise: Gel Electrophoresis

Name: ______________________________

In this experiment, you are testing the hypothesis that the egg from which you isolated DNA

was from the sevruga sturgeon (Acipenser stellatus).

1. If your hypothesis is correct, state the size and number of fragments you would expect to

find in each lane of the gel.

Lane Sample Expected Fragment(s)

1

2

3

4

5

2. If your hypothesis is not correct, identify which samples you expect to differ from those

above, state how they might differ, and explain why.

38

GENETIC TRANSFORMATION OF BACTERIA

WITH THE pBLU PLASMID

Adapted from: Rapoza, M. and H. Kreuzer. Transformations. 2004. Carolina Biological

Supply Company. U.S.A.

BIO 224

Genetics

Fall 2013

Contents General Notes…………………………………………………………………………… 2

Lab 1: Overview of Project and Transformation of Escherichia coli…………………… 3

Lab 2: Transformation of Acinetobacter baylyi………………………………………… 10

Lab 3: Interpreting the Transformation Results…………………………… ………………. 17

General Notes The steps should be followed exactly for each procedure in this manual. You must read

through all of the steps before beginning any of the procedures. If you have questions about a

procedure, you should ask before you begin the procedure.

You must follow all safety precautions described in the procedures. Failure to do so will

result in dismissal from the lab.

39

Lab 1: Overview of Project and Transformation of Escherichia

coli

Objectives 1. Practice sterile technique while working with bacteria.

2. Define transformation.

3. Define bacterial competence.

4. Define plasmid.

5. Define antibiotic resistance.

6. Regarding bacterial transformation:

a. Develop a hypothesis to explain the effect of the pBLU plasmid on Escherichia coli.

b. Carry out a set of experiments to test the above hypothesis.

c. Identify the dependent and independent variables in the experiment.

d. Identify and explain the controls in the experiment.

e. Make specific predictions that follow from the hypothesis.

f. Collect data and summarize it in graphical form.

g. Interpret data and draw appropriate conclusions from the experiment.

h. Identify errors in the experiment.

Before Lab 1. Watch the following animations on bacterial transformation:

http://highered.mcgraw-hill.com/sites/007352543x/student_view0/chapter20/

bacterial_transformation.html

2. Read the lab handout on bacterial transformation.

3. Complete the pre-lab exercise on bacterial transformation (due before lab on Tuesday).

Introduction Genetic transformation is the uptake and expression of genetic material by a living cell.

Bacterial and archaeal cells can naturally undergo transformation, by taking up DNA from

their surroundings. This process allows them to gain new genes that may provide them an

advantage in certain environments. Humans use biotechnology to transform other organisms

for a variety of purposes. For example, when genes coding for insulin or antiviral drugs are

inserted into a few bacteria, and every time those bacteria reproduce, the foreign genes are

reproduced as well. The billions of offspring bacteria become a “factory” for the production

of the protein.

One method for inserting foreign genes into a host organism is through use of a vector. In

biotechnological terms, a vector is a molecule that carries the DNA into the host. Plasmids

are common vectors of prokaryotic DNA. A plasmid (shown in Figure 1 below) is a small,

extra-chromosomal sequence of DNA found in many bacteria and archaea. A plasmid carries

its own genes, but those genes are typically not critical for basic survival and reproduction of

the bacterium. However, if a plasmid contains certain genes, such as those for antibiotic

resistance, then the cell may be able to survive in a new or harsh environment.

40

Figure 1. Bacterial cell containing a plasmid

Competence refers to the tendency of bacteria to take up DNA from their environment.

Different species or strains of bacteria vary in their competence. Highly competent species

will take up DNA relatively easily, while non-competent species will not take up DNA. Even

if a bacterial cell is able to take up DNA, it may not necessarily be able to use, or express, the

genes that are found on that DNA.

The plasmid you will use in this set of experiments is called the pBLU plasmid, which has

been genetically modified to contain two genes of interest (See Figure 2). The first is a gene

for antibiotic resistance. Antibiotics are substances that inhibit the growth and reproduction

of bacteria. Non-resistant bacteria cannot grow or reproduce if exposed to an antibiotic.

However, if a bacterial cell is resistant to antibiotics, then that cell can grow and reproduce in

the presence of the antibiotic. The gene on the pBLU plasmid that confers resistance to the

antibiotic ampicillin is known as ampr (which codes for beta-lactamase, an enzyme that

breaks down ampicillin). Including this gene on the plasmid ensures that the bacteria that

grow in the presence of the antibiotic are only bacteria that contain the plasmid.

The second gene of interest on the plasmid codes for the enzyme β-galactosidase. This

enzyme allows the bacteria to break down the disaccharide lactose. The enzyme will also

break down other types of substances, including the chemical 5-bromo-4-chloro-3-indolyl-β-

D-galactoside (more commonly known as X-gal.) When X-gal is cleaved by β-galactosidase,

one of the products is a dark blue. Therefore, the activity of the β-galactosidase gene can be

detected by the presence of dark blue colonies. If the colonies do not produce β-

galactosidase, then they will be whitish in color.

Figure 2. pBLU plasmid with β-galactosidase and ampr genes

41

Overview The goal of this series of laboratory exercises is to determine the effect of exposing two

different species of bacteria to the pBLU plasmid. Specifically, students will expose

Escherichia coli and Acinetobacter baylyi to the plasmid to determine if they will undergo

transformation. E. coli is a moderately competent species, which naturally contains the gene

for β-galactosidase (in this experiment, we will use a strain that has had the gene “knocked

out”). A. baylyi is known to be a highly competent species that does not naturally contain the

β-galactosidase gene. Do you think that the degree of competence will determine the ability

of the bacteria to be transformed? Do you think that the natural occurrence of the β-

galactosidase gene in a species will determine the ability of the bacteria to be transformed?

Write a testable hypothesis that answers these questions, below:

Lab 1: Transformation of E. coli

In this lab, you will expose E. coli to the pBLU plasmid, and then culture the bacteria onto

nutrient-rich media to grow. The bacteria will be grown on media that contain a combination

of nutrients, ampicillin, and X-gal.

Lab 2: Transformation of A. baylyi

In this lab, you will expose A. baylyi to the pBLU plasmid, and then culture the bacteria onto

nutrient-rich media to grow. The bacteria will be grown on media that contain a combination

of nutrients, ampicillin, and X-gal.

Lab 3: Interpretation of Results

In this final lab, you will summarize, graph, and interpret the results of exposing the two

species of bacteria to the pBLU plasmid.

42

Transformation of E. coli

Background The strain of E. coli that is used in this experiment has had the β-galactosidase gene “knocked

out” or made non-functional. These bacteria can only use nutrients other than lactose (e.g.

glucose) to survive. If these bacteria are able to undergo transformation when exposed to the

pBLU plasmid, then they will be able to express the β-galactosidase gene and the ampicillin

resistance gene.

This week you will do the transformation of E. coli. Next week, you will collect the data for

the experiment, and also do the transformation for A. baylyi.

Lab Safety 1. Wear closed-toed shoes and secure hair and loose clothing.

2. Wear gloves throughout the procedure.

3. Sterilize your workbench and wash your hands before and after the experiment.

4. Dispose of contaminated materials in the labeled disposal containers only.

5. Keep all materials away from your mouth and nose.

Materials Per Group

Quantity Item

2 sterile microfuge tubes

1 floating microfuge tube rack

1 1,000 μL pipette

1 box sterile pipette tips

4 LB nutrient agar plates: (1 LB; 2 LB/Amp; 2 LB/Amp/Xgal; 1

LB/Xgal)

4 sterile, disposable inoculating loops

1 tube sterile CaCl2 on ice

1 tube sterile LB broth at room temperature

1 permanent marker, fine

1 bottle ethanol

1 small bucket of ice

1 150 mL beaker as disposal container for pipettes and tubes

For Class

Quantity Item

1 water bath, 42°C

1 bottle pBLU plasmid

test tube tongs

43

Procedure 1. Sterilize your work station with ethanol.

2. Wear gloves throughout the procedure.

3. Use sterile technique at all times. This prevents microbes from the environment (the air,

your hands, the table top) from contaminating your tools or agar plates. Some tips for

sterile technique:

A. The tip of your inoculating loop should never come into contact with any surface other

than the bacteria and the media. Remove sterile loops from the packaging by the non-

loop end. Placed used loops into disposal containers.

B. Do not touch or have any contact with the edges of the test tubes. Do not leave your

petri dishes open to the air.

4. Obtain and label on the top one microfuge tube “+p” and a second tube “-p”. Put your

initials on the sides of both tubes. (Plasmid DNA will be added to the “+p” tube and no

plasmid DNA will be added to the “-p” tube.)

5. Use the micropipette to add 250 μL of ice-cold calcium chloride to each tube.

6. Place both tubes on ice.

7. Use a sterile inoculating loop to transfer 1 colony of E. coli from the starter plate to the

tube labeled “+p”. (Be sure not to pick up any of the agar from the plate.) Vigorously spin

the loop in the solution to ensure the cells have been dislodged.

8. Immediately suspend the cells in solution by pipetting up and down five times, using a new

pipette tip. The solution should appear cloudy, with no visible clumps of cells. Return the

tube to ice.

9. Repeat steps 7 and 8 for the tube marked “-p”. Be sure to use a new inoculating loop and

pipette tip.

10. Use a sterile inoculating loop to add one loopful of plasmid DNA to the +plasmid tube.

(When the DNA solution forms a bubble across the loop opening, its volume is 10 μL.)

Immerse the loopful of plasmid DNA directly into the cell suspension and spin the loop to

mix the DNA with the cells.

11. Incubate both tubes on ice for 15 minutes.

12. While the tubes are incubating, label your media plates with the date and your lab group

name/initials, as described below.

a. Label one LB/Amp, and one LB/Amp/X-gal with “+plasmid”. These are the

experimental plates.

44

b. Label one LB, one LB/Amp, one LB/X-gal, and one LB/Amp/X-gal with “-plasmid”.

These are the control plates.

13. After the 15-minute incubation on ice, “heat shock” the cells. Remove both tubes directly

from ice and immediately immerse them in the 42°C water bath for 90 seconds. Gently

agitate the tubes while they are in the water bath.

14. Return both the tubes immediately to ice for 1 or more minutes.

15. Use a micropipette to add 250 μL of Luria broth (LB) to each tube. Gently tap the tubes

with your finger to mix the LB with the cell suspension. Use a new pipette tip for each

transfer.

16. Place the tubes in a test-tube rack at room temperature for a 15-minute recovery and

growth period.

17. Now you will remove some cells from each transformation tube and spread them on the

plates. Cells from the –plasmid tube should be spread on the –plasmid plates, and cells

from the +plasmid tube should be spread on the +plasmid plates.

18. Use a micropipette to transfer 100 μL of cells from the –plasmid transformation tube to

each appropriate plate.

19. Use a sterile inoculating loop to spread the cells around the entire surface of the plate.

20. When you finish spreading, let the plates rest for several minutes to allow the

cellsuspensions to become absorbed into the agar.

21. Repeat steps 16 – 19 for the + plasmid plates.

22. Wrap the plates together with tape and place the plates upside down at room temperature.

They will be allowed to incubate for a few days at room temperature, and then stored in

the refrigerator until the following week.

23. Dispose of all used pipettes and tubes, and clean your work station with ethanol.

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Lab 2: Transformation of Acinetobacter baylyi

Objectives 1. Practice sterile technique while working with bacteria.

2. Regarding bacterial transformation:

a. Develop a hypothesis to explain the effect of the pBLU plasmid on Acinetobacter

baylyi.

b. Carry out a set of experiments to test the above hypothesis.

c. Identify the dependent and independent variables in the experiment.

d. Identify and explain the controls in the experiment.

e. Make specific predictions that follow from the hypothesis.

f. Collect data and summarize it in graphical form.

g. Interpret data and draw appropriate conclusions from the experiment.

h. Identify errors in the experiment.

Before Lab 2 1. Read the lab handout on bacterial transformation of A. baylyi.

2. Complete the pre-lab exercise on bacterial transformation (due before lab on Tuesday).

Lab Safety 1. Wear closed-toed shoes and secure hair and loose clothing.

2. Wear gloves throughout the procedure.

3. Sterilize your workbench and wash your hands before and after the experiment.

4. Dispose of contaminated materials in the labeled disposal containers only.

5. Keep all materials away from your mouth and nose.

6. Use caution when using flames.

Background Acinetobacter is not known to carry the β-galactosidase gene in nature. These bacteria use

other genes to break down lactose. If these bacteria are able to undergo transformation when

exposed to the pBLU plasmid, then they will be able to express the β-galactosidase gene and

the ampicillin resistance gene.

A. baylyi ADP1 is known to be highly competent, and is able to take up foreign DNA easily

when the population is in exponential growth phase. For this reason, the procedure for

transformation is much simpler than that for E. coli.

This week you will collect the data from the transformation experiment on E. coli, and also do

the transformation for A. baylyi. Next week, you will collect the data for the A. baylyi

experiment and interpret all of your data.

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Materials Per Group

Quantity Item

2 sterile microfuge tubes

1 floating microfuge tube rack

1 1,000 μL pipette

1 box sterile pipette tips

4 LB nutrient agar plates: (1 LB; 2 LB/Amp; 2 LB/Amp/Xgal; 1 LB/Xgal)

4 sterile, disposable inoculating loops

1 tube sterile LB broth at room temperature

1 permanent marker, fine

1 bottle ethanol

1 150 mL beaker as disposal container for pipettes and tubes

6 E.coli transformation plates from the previous week

For Class

Quantity Item

2 tubes A. baylyi, in broth in exponential growth phase

E. coli Transformation Results 1. Obtain your agar plates from the previous week. Examine the plates for bacterial growth

and for the presence of blue colonies. Record your data on the returned post-lab exercise

from the previous week.

A. baylyi Transformation Procedure 1. Sterilize your work station with ethanol.

2. Wear gloves throughout the procedure.

3. Use sterile technique at all times. This prevents microbes from the environment (the air,

your hands, the table top) from contaminating your tools or agar plates. Some tips for

sterile technique:

A. The tip of your inoculating loop should never come into contact with any surface other

than the bacteria and the media. Remove sterile loops from the packaging by the non-

loop end. Placed used loops into disposal containers.

B. Do not touch or have any contact with the edges of the test tubes. Do not leave your

petri dishes open to the air.

C. To keep microbes from falling into open culture tubes, you need to flame the mouth of

the tube quickly after opening. Flame the mouth of the tube before and after taking a

sample. Do not set the lid down on the counter at any time- keep it held in the crook

of your pinky finger, face down.

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4. Obtain and label on the top one microfuge tube “+p” and a second tube “-p”. Put your

initials on the sides of both tubes. (Plasmid DNA will be added to the “+p” tube and no

plasmid DNA will be added to the “-p” tube.)

5. Obtain a culture of A. baylyi in LB broth. This culture is at exponential growth phase,

meaning that the cells have been dividing rapidly. This is the phase at which A. baylyi is

most able to undergo transformation.

6. Transfer 200 μL of the liquid culture to each microfuge tube (“+p” and “-p”).

7. Use a sterile inoculating loop to add one loopful of plasmid DNA to the +plasmid tube.

(When the DNA solution forms a bubble across the loop opening, its volume is 10 μL.)

Immerse the loopful of plasmid DNA directly into the cell suspension and spin the loop to

mix the DNA with the cells.

8. Use a micropipette to add 250 μL of Luria broth (LB) to each tube. Gently tap the tubes

with your finger to mix the LB with the cell suspension. Use a new pipette tip for each

transfer.

9. Place the tubes in a test-tube rack at room temperature for a 15-minute recovery and

growth period.

10. While the tubes are incubating, label your media plates with the date and your lab group

name/initials, as described below.

a. Label one LB/Amp, and one LB/Amp/X-gal with “+plasmid”. These are the

experimental plates.

b. Label one LB, one LB/Amp, one LB/X-gal, and one LB/Amp/X-gal with “-plasmid”.

These are the control plates.

11. Now you will remove some cells from each transformation tube and spread them on the

plates. Cells from the –plasmid tube should be spread on the –plasmid plates, and cells

from the +plasmid tube should be spread on the +plasmid plates.

12. Use a micropipette to transfer 100 μL of cells from the –plasmid transformation tube to

each appropriate plate.

13. Use a sterile inoculating loop to spread the cells around the entire surface of the plate.

14. When you finish spreading, let the plates rest for several minutes to allow the cell

suspensions to become absorbed into the agar.

15. Repeat steps 11 – 14 for the + plasmid plates.

16. Wrap the plates together with tape and place the plates upside down at room temperature.

They will be allowed to incubate for a few days at room temperature, and then stored in

the refrigerator until the following week.

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17. Dispose of all used pipettes and tubes, and clean your work station with ethanol.

18. Complete the Analysis below.

Analysis

1. Which plates showed bacterial growth? Which plates had bacteria that glowed?

2. Which of the plates were controls, and why?

3. Which bacteria underwent transformation, and how do you know?

4. Were your predictions met? Was your hypothesis supported?

5. What were the sources of error in your experiment?

6. How could the uptake of plasmids in a natural environment be beneficial to bacteria?