1431470 Bacterial Trans TG InHousePrinting · expression • To explore the concept of phenotype...

Neo/SciTeacher’s Guide

4 Lab Stations #14308878 Lab Stations #1431271

Bacterial Transformation

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Lab 8

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Teacher’s Guide

Students then have the opportunity to design and conduct individual experiments to induce mutations with observable phenotypes, or modify the transformation process to increase or decrease the uptake of plasmid by manipulating environmental conditions thereby exploring the mechanism of transformation in more depth.

CORE CONCEPTSBig Idea 3: Living systems store, retrieve, transmit, and respond to information essential to life processes. • Genetic information provides for continuity of life,

and, in most cases, this information is passed from parent to offspring via DNA.

• When the DNA of a cell changes, the RNAs and proteins they produce often change, which in turn changes how that cell functions.

• DNA inside a cell can change several ways. It can be mutated, either spontaneously or after the DNA replication machinery makes an error.

• Biotechnologists may cause an intentional mutation in a cell’s own DNA.

• One common technology, bacterial plasmid-based genetic transformation, enables students to manipulate genetic information in a laboratory setting to understand more fully how DNA operates.

Lac Z

Amp

2,686 BP

AmpR = Ampicillin Resistance geneLac Z = Gene for Galactosidase

ALIGNMENT TO AP® CURRICULUM FRAMEWORK

This investigation utilizes the concepts of Big Idea 3, those being storage, retrieval, transmission of genetic information, and responding to information. It allows students to have hands on experience with genetic engineering and to perform transformation experiments with E. coli bacteria.

ENDURING UNDERSTANDINGS: • 1A2: Natural selection acts on phenotypic variations in

populations. • 1C3: Populations of organisms continue to evolve. • 3A1: DNA, and sometimes RNA, is the primary source

of heritable information. • 3B1: Gene regulation results in differential gene

expression. • 3C1: Changes in genotype can result in changes in

phenotype. • 3C2: Biological systems have multiple processes that

increase genetic variation.

Additional information on Curriculum Alignment may be found at Correlation Station: http://www.freyscientific.com/SupplementalCurriculum/InquiryInvestigations/CorrelationStation/tabid/395/Default.aspx

LAB OVERIEW

Copyright © Delta Education LLC. A member of the School Specialty Family. All rights reserved.

This guided and open investigation provides an introduction to some of the techniques used in the field of genetic engineering. In the model experiment, students will perform a procedure that will transform a special strain of E. coli bacteria using a plasmid vector to express new genetic information that will confer ampicillin antibiotic resistance to the E. coli. The plasmid that will be used to transform the bacteria in this investigation is pUC18 and occurs naturally in E. coli. Transformed bacteria cells will grow in the presence of the antibiotic while bacteria cells that did not take up the plasmid, will not grow. Students will use mathematical calculations to determine the efficiency of the transformation process.

LEARNING OBJECTIVES • To demonstrate the universality of DNA and its

expression • To explore the concept of phenotype expression • To explore how genetic information can be transferred

from one organism to another • To investigate the concept of horizontal gene transfer

and the subsequent increase in genetic variation

Figure 1: Plasmid pUC18 A plasmid is a circular dsDNA molecule. This plasmid has been genetically engineered to include a gene for antibiotic resistance to ampicillin (ampR) and a gene (and its promoter) for the enzyme beta-galactosidase (laZ).SAMPLE

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• To explore the relationship between gene expression and certain environmental factors

• To work collaboratively to define investigationproblems

TEACHING INQUIRY

The instructional strategies that underlie these investigations focus on exposing students to example ‘model’ investigations where they learn experimental protocol and investigative techniques. This is followed by student-directed experimental design investigations (independent inquiry paths) where students develop and test hypotheses, conduct investigations, analyze data, and communicate results to you and to their peers. Herron (1971) identifies four levels of inquiry1: • Confirmation - known science content principles are

confirmed by students through experimental action • Structured - students investigate a teacher-

presented question through a prescribed procedure • Guided - students investigate a teacher-presented

question using student-designed/selected procedures

• Open - students investigate topic-related questions that are formulated through student-designed/selected procedures

EXPERIMENTAL DESIGN CONSIDERATIONS To help students understand the material they will be expected to apply in this activity, be sure your students can understand and discuss the following concepts before they start this lab activity. • Do all plasmids contain the same information? • Are all plasmids the same size? • Is there a difference between genetically engineered

and naturally occurring plasmid DNA? • Do all plasmids transform bacteria with the same

efficiency? • Does it matter what type of bacteria takes up a

plasmid? • Can a bacterial cell take up more than one type of

plasmid at the same time? • How could you determine the maximum expression of

a transformed plasmid? • Do plasmids just confer antibiotic resistance or is there

another survival advantage? • Can bacteria express proteins from different organisms?

1Herron, M.D. (1971). The Nature of Scientifi c Inquiry, School Review, 79(2), 171-212.

MODEL EXPERIMENT

This kit contains enough materials for either 4 or 8 groups of students (4 per group) to perform the model experiment. Students will perform a bacterial transformation using E. coli strain JM101 with the DNA plasmid pUC18. Students will be given step-by-step instructions on how to prepare competent bacterial cells which will take up the plasmid. Using a selective media plate, students will identify transformed colonies and calculate the efficiency of the procedure.

It is essential that each student have a good understanding of the techniques involved in manipulation of the bacteria as well as the biochemical and cellular processes involved in the expression of the β-lactamase gene of the plasmid which confers the antibiotic resistance necessary for the bacteria to survive in the presence of ampicillin. Given this knowledge, students should be able to expand on this procedure to investigate other variables in the process.

INDEPENDENT INQUIRY PATHWAYS

The Independent Investigation Inquiry Paths are broken up into two sections. Part A lists possible experiments that can be completed with the materials that are already given to you in the kit. Essentially you have enough materials to complete the model experiment twice.

Part B experiments would require the purchase of additional materials. You will need to determine which (if any) of the experiments you and your students can complete given your constraints and then direct the students to the experiments they can consider doing. There is additional preparation work for you (preparing plates, etc.) that would need to be done ahead of time. You can decide if you want to limit the options of students and perform the experiments right away, or have the students design their experiments, and then prepare and order any additional materials you need. You can consult the end of this teacher guide for a list of what materials you can order to do these Independent Inquiry Paths.

It is recommended that you have students choose one Independent Inquiry Path to complete from either Part A or Part B. They should be able to design their experiment, carry out the experiment, and analyze the results in approximately three class periods.

At the conclusion of the independent investigations, your students can create mini-posters that showcase the results of their Independent Investigation experiments or provide a formal lab report. Students can receive instruction on how to complete a mini-poster session from the provided Resource Series presentation and we have included a sample rubric for a formal lab report.


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KEEPING A LABORATORY NOTEBOOK

A lab notebook allows your students to organize their work for use in a later, formal report. It should contain: • Work group members • Primary question (stated problem) for investigation • Background observations and contextual information • Hypothesis and rationale for the investigation • Notes on Procedure / Experimental Design —

strategies for testing hypothesis, using appropriate controls and variables

• Materials required • Safety issues (or specific cautions) • Procedure in sufficient detail so that another student

group could replicate team results • Results, including graphs, tables, drawings or

diagrams, and statistical analysis • Conclusion and discussion — Was the hypothesis

supported? What additional questions remain for further investigation?

• Citations for sources found through library or web research

INVESTIGATION MATERIALS AND PREPARATION

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8-Lab Stations

Quantity Item Description 1 tube E. coli MicroLIVE culture 40 ea Sterile 1mL Micropipets 32 ea Sterile 2mL microfuge tubes (2 pkg of 16) 200μL pUC18 solution (0.005μg/μL) 30mL 0.05M CaCl2 40 ea Sterile inoculating loops 40 ea Spreading rods 10mL Luria broth (2-5mL vials) 1100mL Luria broth agar (4-275mL bottles) 2 vial Ampicillin (0.02g dry powder) 33 ea Petri plates, sterile

MATERIALS NEEDED BUT NOT SUPPLIED

(Per Student) Apron Gloves Goggles

(Per Group) Marker Clear tape Digital or fixed volume micropipette

(with at least 5 tips)Microfuge tube rackContainer with ice

(Per Class) Hot plate with large beaker of water at 42°C Incubator (37°C) Rubbing alcohol Flame source Microwave 5% Household bleach solution


KIT MATERIALS & NEEDED EQUIPMENT 4-Lab Stations

Quantity Item Description 1 tube E. coli MicroLIVE culture 20 ea Sterile 1mL micropipets 16 ea Sterile 2mL microfuge tubes 100μL pUC18 solution (0.005μg/μL) 30mL 0.05M CaCl2 20 ea Sterile inoculating loops 20 ea Spreading rods 5mL Luria broth 550mL Luria broth agar (2-275mL bottles) 1 vial Ampicillin (0.02g dry powder) 33 ea Petri plates, sterile

OPTIONAL MATERIALS FOR INDEPENDENT INQUIRY INVESTIGATION: See References and Resources for description and ordering informationSAMPLE

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PRE-LAB PREPARATION

Pre-lab Preparation: 1.5 – 2 hours approximately 48 hours before the lab.

Note: You will need to do similar preparation to prepare additional plates for the students for their Independent Inquiry Path Investigations.

Pouring the agar plates:

Caution: Wear heat protective gloves when pouring hot agar. 1. Divide the petri plates into one group of 17 to be

poured with Luria broth agar, and another group of 16 to be poured with Luria broth agar containing ampicillin. Be sure to keep the two piles of plates separate and in clearly marked groups. One of the plain Luria broth agar plates will be used for culturing E. coli cells prior to the laboratory exercise. The remaining petri plates will be distributed to the class groups (two of each type of plate per group).

2. Open the cap on the Luria broth agar bottles and place them in a microwave. Heat the agar in 1-minute intervals, until it is completely liquified.

3. Remove the agar bottles from the microwave and allow them to cool down for several minutes.

4. Open a petri plate lid as little as possible and pour enough agar (approximately 15 mL) into the plate to cover the bottom. Close the lid. Repeat with 16 more petri plates. These are the plain Luria broth plates.

5. When the second Luria broth bottle has cooled enough for you to hold in your hand (but before it sets), pour in the ampicillin powder provided with the kit. Gently invert the bottle a few times to mix the ampicillin with the agar—DO NOT SHAKE.

6. Repeat Steps 2-4 with the Luria broth agar with ampicillin and the remaining 16 Petri plates.

7. Allow all the plates to harden undisturbed. Refrigerate upside down until needed.

Reconstituting the MicroLIVE™ E. coli culture:The system included consists of a sterilized dispensing tube containing two crushable ampules. The larger ampule contains a special reconstituting medium. The smaller ampule contains the inactive bacterial organism.

To reconstitute the culture:

1. Hold the dispensing tube between the fingers of both hands. Place your thumbs on the label markings. Gently exert pressure between your thumbs and forefingers, breaking both inner ampules.


Always follow proper lab safety and aseptic technique protocols. Require the students to wear protective gloves, goggles, and a lab apron when working with bacteria. Remind the students that once their petri plates have been inoculated with bacteria, they must be sealed with clear tape and not opened.Wear heat protective gloves when pouring hot agar. Remind the students not to touch their faces or mouths with their hands. They should wash their lab table and their hands immediately after handling bacteria. Students should keep their work area clean and dispose of all materials as instructed.Treat all bacterial organisms as if they are pathogens. Practice aseptic technique and good personal hygiene. If a spill occurs, put on safety goggles, apron, and disposable gloves and wipe the area with dilute (5%, 1:20) bleach solution. Use tape to seal the dispensing tip of the MicroLIVE culture prior to disposal. Autoclaving at 121°C, 15 psi, for at least 15 minutes is still the best method of disposing of contaminated microbial culture materials. If an autoclave is not available, apply a small amount—just enough to cover the culture surface—of dilute bleach solution (5%, 1:20). Seal with tape and dispose.At the end of the lab exercise, collect all petri plates and any contaminated items such as inoculating loops and spreading rods, etc. Prior to disposal, sterilize all contaminated materials by immersing them in a 5% bleach solution for about 1 hour. The work area should be decontaminated by wiping it with either a bleach solution or a 95% ethanol solution.

SAFETY & DISPOSAL

TIME REQUIREMENTS (6-7 DAYS) Pre-lab Assessment: 60 minutes (1 class period or

homework)Model Experiment: 60 minutes (1 class period)Analysis of Experimental Results: 60 minutes (1 class period)Experimental Design: 60 minutes (1 class period)Independent Investigation Experiment: 60 minutes (1 class period)Analysis of Experimental Results: 60 minutes (1 class period)Presentation: 60 minutes (1 class period)

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reinforce these necessary research skills by having the students perform the following tasks: • Transforming bacterial cells with plasmid DNA • Streaking transformed and non-transformed bacteria

onto LB Plates • Streaking transformed and non-transformed bacteria

onto antibiotic containing LB Plates • Proper disposal of contaminated experimental

materials and solutions • Use of sterile technique to culture microorganisms • Measuring and monitoring temperature in °C • Using a metric prefix to indicate the proper

magnitude when defining volume and mass in liters and grams

• Using pipettes and/or pipettman to transfer and deliver specific volumes of material to sterile vials

• Applying quantitative mathematical skills when calculating transformation efficiency

An important part of becoming a scientist is to learn to keep clear, concise, and accurate laboratory notes. A lab notebook should demonstrate originality and reflection while serving as a record of the investigator’s work. A good laboratory notebook record should include: • Primary question (problem) / hypothesis • Notes: contextual information; procedure and/or

experimental design • Data tables • Drawings / illustrations • Observation recordings / results • Analysis / conclusions

QUANTITATIVE SKILLS RESOURCES CD-ROMThe CD-ROM provided in Neo/SCI’s Investigations for AP® Biology includes a Quantitative Skills Resource that contains PowerPoint presentations and is available in PDF format (with Presenter’s Notes) that can be emailed to students. The PowerPoint is also available as free app that plays the presentation as a movie on an iPad, iPod, and Android operating systems. Refer to HYPERLINK “http://www.schoolspecialty.com” www.schoolspecialty.com for a link to iTunes to download the free app that students can use on their mobile devices. Topics include: Statistical Analysis & Graphing, Mendelian Genetics, EXCEL Spreadsheet Operations, Mathematical Modeling & BioInformatics, Mini-Poster Sessions, and Lab Prep Skills.

2. Shake to mix the reconstitution medium and bacteria pellet. Do so by holding the dispensing tube at the dropper end and shaking the tube like a fever thermometer about 6 to 8 times.

3. Allow the dispensing tube to stand for approximately 15-20 minutes to ensure thorough reconstitution of the culture.

To inoculate a plate culture:

1. Hold the dispensing tube near the tip and exert gentle pressure to dispense drops of living cells from the tip. Once dispensing has occurred, avoid contact of the dispensing tip with any other surface. Should contact occur, simply wipe the surface with 70% rubbing alcohol to resterilize.

2. Using aseptic technique, gently dispense one or two drops onto the surface of a partially uncovered Luria broth agar plate. Close the plate and gently tilt it, allowing the drops to spread out. You may also use a sterile applicator swab to spread the culture. Repeat with the other E. coli growth plate. Incubate at 37°C until 2-3mm diameter colonies appear on the agar surface (24-48 hours prior to use).

HELPFUL HINTS / TECHNIQUES Satellite Colonies- smaller, less abundant colonies surrounding the larger, more prominent colonies on the agar plates containing ampicillin may be observed while collecting data for the results. These satellite colonies form when transformed cells release beta-lactamase (the enzyme encoded by the plasmid that degrades ampicillin) into the surrounding medium. Non-transformed bacteria can then survive and grow.Bacterial cells made competent in a properly equipped science lab or purchased from a commercially available source will yield 108-109 transformed colonies per microgram of plasmid. Competent cells obtained from this preparation will yield approximately 103-104 transformed colonies per microgram of plasmid.

STUDENT SKILLS DEVELOPMENT

Students need to develop skills in communication, teamwork, critical thinking, accurate and safe manipulation and disposal of biological materials and equipment. This investigation can help foster and


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GETTING READY

Prior to beginning the model experiment, students should read through, or view the PowerPoint presentation Lab Skills to thoroughly understand what is involved in Experimental Design, Defining a Problem, Keeping a Laboratory Notebook, and Identifying a Variable.

Students need to be familiar with the techniques involved in manipulating DNA, bacteria and to have a reasonable understanding of what is involved in the model experiment. They should be very clear on what to do and what not to do when following a procedure which requires sterile technique so as not to introduce a contaminating microorganism.

The tutorial on Bacterial Transformation presents the general procedure used to transform bacteria:http://www.youtube.com/results?search_query=bacterial+transformation

After watching the tutorial, you may also consider having your students practice, under your supervision, the following activities as a “dry run” prior to beginning the lab:

• Using an inoculating loop to streak a plate. This does not have to be done with the bacteria, but you should confirm that your students are proficient and careful enough not to gouge the agar with the loop.

• Using a pipette to transfer and deliver specific volumes of liquid, making certain that the students comprehend the gradations and volume markings on the exterior surface.

THE INVESTIGATION Bacterial Transformation – Ampicillin Resistance THINK ABOUT IT...Have your students answer these questions in their lab notebook before completing the Investigation.

• Explain the process of transformation.

• What are plasmids? How are they transferred?

• What is genetic engineering? How is it used?

• List some examples of genes found in genetically engineered plasmids.

• Can bacteria express proteins from different organisms?

• Can a bacterial cell take up more than one type of plasmid?

(Per Student) Gloves Apron Goggles

(Per Group) Marker 2 2-mL Sterile microfuge tubes Microfuge tube rack 500μL 0.05 M CaCl₂ (on ice) 1 Sterile inoculating loop 10μL pUC18 solution 2 Luria broth agar plates 2 Luria broth agar with ampicillin plates 500μL Luria broth 1 Spreading rods 1 Digital or fixed volume micropipet

(with at least 5 tips)Clear tape

(Per Class) E. coli culture plate Ice bath 42°C Water bath Rubbing alcohol Flame source Incubator set at 37°C

STEP 1

Each student group should obtain two poured Luria broth plates, and label one of the plates “LB+”, and the other plate “LB-”.

STEP 2

Each student group should obtain two poured Luria broth with ampicillin plates, and label one of the plates “LB/Amp+”, and the other plate “LB/Amp-”.

STEP 3

Each student group should obtain two sterile 2-mL microfuge tubes, and label one of the tubes “+”, and the other tube “-”.

WHAT YOU NEED

WHAT TO DO


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STEP 4

The students should use one sterile, plastic 1-mL micropipet, to add 250 μL of ice cold 0.05 M CaCl₂ to each of the sterile microfuge tubes.

STEP 5

The students should use a sterile, plastic inoculating loop to add 2-3 E. coli colonies to each of the two 2mL microfuge tubes. Caution them to try not to gouge the agar that the E. coli are growing on as no agar should be introduced to the tubes. They should tap the inoculating loop firmly against the side of each tube to ensure that a colony is introduced to each tube, and discard the inoculating loop according to your instructions.

STEP 6

The students should use the micropipet from Step 4 to suspend the cells in the CaCl₂ in the tubes. They may accomplish this by alternately drawing the solution from the tube into the micropipet and emptying it several times, and then discard the micropipet as you have directed.

STEP 7

The students should use a digital or fixed volume micropipet to introduce the antibiotic-resistance plasmid by adding 10μL of the pUC18 solution into the “+” tube, and mix the contents by tapping on the tube.

STEP 8

The students should place both microfuge tubes directly in the ice container for a total of 15 minutes.

STEP 9

The students must heat shock the bacterial cells by placing both tubes in a water bath at 42°C for a total of 90 seconds.

STEP 10

The students should return both tubes immediately to the ice container, and allow them to remain there for 2 minutes.

STEP 11

Student should use one sterile, plastic 1-mL micropipet to add 250 μL of Luria broth to each of the microfuge tubes and tap on each tube to mix the contents. They should then place the tubes in the microfuge tube rack at room temperature and discard the micropipet as you have directed.

STEP 12

The students should use a digital or fixed volume micropipet to draw 100 μL of “+” cells and dispense them onto the “LB+” plate. They should place 100 μL of “+” cells on the “LB/Amp+” plate also.

STEP 13

The students should sterilize the spreading rod by dipping it in alcohol and flaming it before use. They should spread the “+” cells across each of the two “+” plates. They must cover the plates immediately and place them aside to set for a few minutes. The students should then resterilize the spreading rod immediately.

STEP 14

The students should use a clean tip on the digital or fixed volume micropipet to draw 100 μL of “-” cells and dispense them onto the “LB-” plate. They should place 100 μL of “-” cells on the “LB/Amp-” plate also.

STEP 15

The students should again sterilize the spreading rod by flaming, and spread the “-” cells across each of the two “-” plates. The students must cover the plates immediately and place them aside to set for a few minutes. They should resterilize the spreading rod as they did previously.

STEP 16

All plates should be taped with clear tape at a few places around the rim, and incubated inverted in a 37°C incubator overnight.

Note: The petri plates should not be completely sealed as this would create anaerobic growth conditions

DATA SUMMARY AND ANALYSIS

Observe each of the incubated plates. Do NOT open them; colonies are visible through the bottom of the plates. Count the total number of colonies present on each plate and enter those values in Data Table 1. If the colonies are too numerous to count (i.e., the colonies have grown into each other), this is called a “lawn.” Record this in Data Table

DATA TABLE 1

Plate Number of Colonies

LB+ Lawn

LB- Lawn

LB/Amp+ 20

LBAmp- 0


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Lab 81–800–526–6689

The total number of antibiotic-resistant bacterial colonies visible on each plate can be used to calculate the transformation efficiency, or the number of antibiotic-resistant colonies/μg pUC18. Perform the following calculations to determine the transformation efficiency.In this procedure, a total volume of 10μL of pUC18 (plasmid) at a concentration of 0.005μg/μL were used. Calculate the total mass of the pUC18 used:Total mass of pUC18 (mass = volume x concentration): 0.05 μg Total volume of cell suspension: 510μL Fraction of the total cell suspension spread on plate (μL spread/total volume): 0.196Mass of pUC18 in the cell suspension (total mass x fraction spread on plate): 0.0098The transformation efficiency is the number of colonies/μg plasmid (# colonies observed/mass of pUC18 in suspension): 2.04 x 10₃ (use scientific notation)

ANALYSIS QUESTIONS

1) Three of the plates were controls. Which three plates were the controls? For each, describe how it was a control.

LB+ and LB- were both positive controls. These two plates demonstrated the degree of bacterial colonization that occurs when antibiotic is not present, regardless of whether the cells were transformed (+) or not (-). LB/Amp- was a negative control. This plate demonstrated the result of attempting to grow non-transformed cells (cells without antibiotic resistance) on a plate with antibiotic present.

2) Make a pairwise comparison of the following plates: LB+ and LB-; LB+ and LB/Amp+; LB- and LB/Amp-; LB/Amp+ and LB/Amp-. Describe the growth on each pair of plates and discuss why the growth appeared as it did.Both LB+ and LB- have a lawn of bacteria on them. Since neither plate on which these bacteria were grown had any antibiotic in it, this amount of growth is to be expected. LB+ and LB/Amp+ differed in that LB+ was covered by a lawn, but LB/Amp+ had about 20 colonies. Growth on the LB/Amp+ plate showed that some of the bacterial cells had been transformed. LB- and LB/Amp- also differed: LB- had a lawn of growth, while the LB/Amp- showed no growth at all. This illustrates the effectiveness of ampicillin against non-transformed bacterial cells. LB/Amp+ and LB/Amp- were different; however, both plates showed the ability of ampicillin to control bacterial growth. None of the cells plated on the LB/Amp- plate were transformed, so all of them were susceptible to the antibiotic. On the LB/Amp+ plate, none of the non-transformed cells were able to form colonies; the transformed cells did grow, indicating the presence of the antibiotic-resistance gene in cells in those colonies.

3) Explain the reason for heat shocking the bacteria.In the plasmid transformation, after the heat shock step, intact plasmid DNA molecules replicate in bacterial host cells which produces multiple copies of a single plasmid within the bacteria.

4) How do you determine whether a bacterium has been transformed?Confirmation of transformation can be verified by use of selective media (e.g. ampicillin, tetracycline) or looking for the novel protein (e.g. green fluorescent protein, blue fluorescent protein)

5) How can you differentiate a satellite colony from a transformed colony?You can streak the suspect colony on a fresh plate. If it survives it was transformed with the plasmid, if not, it was a satellite.

6) What is the reason for culturing the bacteria at 37°C?E. coli flourish in the large intestines of many mammals including humans. Our normal body temperature is 98.6°F or 37°C

7) List the steps in the procedure that require sterile technique or materials.

a) Reconstitution of the E. coli b) Transfer of colonies to the sterile tube to make them

competent. c) Addition of the plasmid to the competent bacterial cells.

8) Explain why sterile technique or materials are necessary in those steps.Sterile technique is required so as not to introduce a foreign microorganism into the experiment which may affect the results.

INDEPENDENT INVESTIGATION INQUIRIES

DESIGNING & CONDUCTING INDEPENDENT INQUIRY PATHS Students are given a list of Part A and Part B ideas for their independent inquiry. You need to decide what (if any) Part B investigations you can allow students to perform. Instruct students what options they have, and then let them start designing an experiment to test the investigation idea.


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2) Compare transformation efficiency using pUC57 (a smaller ampicillin resistant plasmid).

Decreased plasmid size should increase transformation efficiency.

3) Transform the bacteria with pUC18 and pBR322 to determine if the bacteria can take up more than one plasmid at a time.

Students can plate the bacteria on an ampicillin/tetracycline plate (as well as the Luria broth). If any bacteria grow on the amp/tet plate, then they were able to pick up both plasmids.

4) Perform a novel transformation experiment to show that the bacteria produce a protein which is not part of their original genetic complement.

We have listed some examples of plasmids that you can purchase to demonstrate the production of a protein. Transform the bacteria with a plasmid that glows under UV light or produces a colored pigment. This requires genetically engineered bacteria that express an RNA polymerase capable of binding to the 5’ upstream region of the plasmid promoter to transcribe the novel gene.

EVALUATINGSUMMATIVE ASSESSMENT: LABORATORY REPORT OR POSTER SESSION

You may choose various evaluation modalities to assess student comprehension. At a minimum, students should submit their laboratory notebooks to you for review (and possible grading). At a minimum, the following areas should be covered in their notebooks:

- Problem Statement - Background Notes / Questions / Resource References - Materials - Experimental Design (protocol / techniques) - Experimental Data: graphs, results - Conclusions

Students should review the Mini-Poster Sessions presentation and be able to either:- create a mini-poster session and present it to the class OR- write an Overview or Summary statement (as for a

scientific journal) that summarizes their investigational results.

Each Independent Investigation is listed below and expected results are given for each.You will need to make sure that you have enough agar plates prepared for students to use for their chosen experiment. We have provided enough agar and plates for you to make approximately 4 additional plates per group. As the students are planning, let them know how many plates they can use in their inquiry experiment.

Part A:

1) What is the effect of changing the pH of the CaCl₂ solution?

The pH of the CaCl₂ solution has been optimized for this procedure. Altering the pH will decrease the transformation efficiency.

Note: You will need to provide students with a weak acid or base to allow them to alter the pH. The initial pH of the CaCl₂ solution is approximately 5.

2) Determine the upper limit of antibiotic resistance by increasing the concentration of ampicillin in a series of LB plates.

Initial concentration of ampicillin in the LB agar is 20mg/275mL. Increasing the amount of antibiotic to 10x that amount should greatly reduce or eliminate colony formation by any transformed bacteria obtained from the model experiment.

3) What is the effect of changing the length of the heat shock?

The length of the heat shock has been optimized for this procedure. Altering the length of time will decrease the transformation efficiency.

Part B:

The additional materials you need for this section are listed in the optional materials section and suggested ordering information is given at the end of the guide.

1) Compare transformation efficiency using pBR322 (a larger, ampicillin resistant plasmid).

Increased plasmid size should reduce transformation efficiency if all other variables are held constant.


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INFORMATION TABLE 1Lab Report Rubric

Title Page

Title of the labStudent’s nameDateTeacher’s class

5pts

IntroductionBackground informationPurpose for doing the experimentHypothesis

10pts

Materials and

Methods

-ALL Materials used to perform the lab-Procedure as a numbered step-by-step

description on how they performed the experiment

10pts

Results

-Tables -titled, clearly labeled columns-Line Graphs-titled, x & y axes labeled

correctly as dependent and independent variables

-Histograms- titled, axes labeled correctly-Statistical analysis if required-Digital Record (pictures, video)

25pts

Discussion

-Results interpreted and summarized-Source(s) of possible error identified-Analysis of Hypothesis- statement

declaring whether it was or was not supported

-Suggestions for improvement if applicable

25pts

Questions Determine Student knowledge of process 15pts

Cited literature

Internet, text book, etc.Citation Accuracy (footnote, bibliography)

5pts

Language Usage

GrammarPunctuationSpelling

5pts

GOING FURTHER

The background to this investigation asks students to think about several applications of genetic transformation, including genetically modified food and possible ethical, social, or medical issues raised by the manipulation of DNA by biotechnology.• Ask students to discuss why these issues are “issues.”

What questions are posed by genetic engineering?• Students also can respond to the quote from Michael

Crichton’s novel and film Jurassic Park: “Just because science can do something doesn’t mean that it should.”

REFERENCES AND RESOURCES

OPTIONAL MATERIALS FOR INDEPENDENT INVESTIGATION INQUIRIESAvailable at FREY Scientific: http://www.freyscientific.com

Part Number DescriptionH578952 0.5% Hydrochloric Acid Solution (0.1M), 500mLH580194 Sodium Hydroxide Solution (1M), 1 LiterD90-9450 pBR322 Plasmid DNA, 10μg1450948 pUC57 Plasmid DNA, 10μg1450949 pBlue Plasmid DNA, 10μg1450950 pGreen Plasmid DNA, 10μg95-2036 Tetracycline, 1g1450951 T7 Express Competent E.coli, 0.2mL

AP® BIOLOGY RESOURCE App

GET YOUR FREE AP® BIOLOGY App

AP® SKILLS RESOURCE SERIES CD-ROM Statistical Analysis & Graphing

Mendelian Genetics Spreadsheet Basics Bioinformatics Lab Prep Skills Conducting a Mini-Poster Presentation

CORRELATIONS TO ADDITIONAL LABORATORY KITS AND SOFTWAREAvailable at FREY Scientific: http://www.freyscientific.com

NEO/SCI VIRTUAL LABS AND CD’SPart Number Description1366224 iNeo/SCI AP Biology On-line Virtual Lab Series10-1191 Molecular Biology Neo/Lab CD-ROM

If you would like your students to write a formal lab report you can use the rubric below for grading. It may be helpful to give the students the rubric ahead of time so that they are aware of the expectations.


SAMPLE

12

Neo/SCI®

Teacher’s Guide

SMALL SCALE ISOLATION AND AGAROSE GEL PURIFICATION OF PLASMID DNAhttp://www.youtube.com/watch?v=v7w-DisXPM8Describes the process of genetically engineering human insulin and shows a procedure for isolating and purification of plasmid DNA

SUGGESTED WEB RESOURCES

GENETIC TRANSFORMATION OF PLANTS

http://www.scribd.com/doc/43766647/Genetic-Transformation-in-Plants Investigate other means by which DNA is moved into an organism to alter its genetic makeup.

GENETIC TRANSFORMATION OF MAMMALIAN CELLShttp://www.molecularstation.com/transfection/Investigate the use of (cationic) polymers, liposomes and nanoparticles methods of introducing foreign DNA into a eukaryotic cell.

TRANSGENIC ANIMALS-KEY TO HUMAN DISEASE RESEARCH

http://www.ctcase.org/bulletin/11_3/transgenic.html

RECOMBINANT TECHNIQUES FOR PRODUCING TRANSGENIC ANIMALShttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/TransgenicAnimals.html Transgenic animals carry foreign gene(s) that has been stratetically inserted into their chromosomal DNA. In addition to the gene itself, the DNA usually includes other sequences that enable it to be incorporated into the DNA of the host animals cells and to be expressed correctly by those cells.

OTHER RESOURCES

• Dolan DNA Learning Center, Cold Spring Harbor http:www.dnal.org

• Griffith, A.J., Natural plasmids of filamentous fungi. Microbiol. Rev. 1995 December 59 (4) http://www.ncbi.nlm.nih.gov/pubmed/8531891

• Johnson A. Daniel, 40 Inquiry Exercises for the CollegeBiology Lab, NSTA Press, Arlington, VA, 2009 http://phschool.com/science/biology_place

NEO/SCI LABORATORY KITS

Part Number Description20-3283 Bacterial Transformation20-1453 The Biotechnology of Cheesemaking20-2103 Understanding Biotechnology20-3393 Biotechnology in the Classroom

FREY SCIENTIFIC LABORATORY KITS

Part Number Description1278457 Biotechnology Techniques Curriculum Module1278382 Biotechnology Applications Curriculum Module1278401 Learning About DNA and Biotechnology Techniques1278436 Learning About Genetically Modified Plants

1278421 Learning Restriction Site Mapping of DNA

AP COLLEGE BOARD LAB MANUAL REFERENCES

Background Information/ Prelab Activities• The University of Arizona Biology Project http://www.

biology.arizona.edu/• Curriculum Module, AP Biology: From Gene to Protein-

A Historical Perspective, College Board, 2010

YOUTUBE PICKS BACTERIAL TRANSFORMATION http://www.youtube.com/results?search_query=bacterial+transformationPresents the general procedure used to transform bacteria

AP BIOLOGY LAB 6: MOLECULAR BIOLOGY

http://www.youtube.com/watch?v=OZyFX9megs8Paul Andersen explains the process of transformation using the pGLO plasmid to produce glowing E. coli bacteria. He then describes how you can use restriction enzymes and the process of gel electrophoresis to cut and separate DNA.


SAMPLE

1431470 Bacterial Trans TG InHousePrinting · expression • To explore the concept of phenotype...

Documents

Transcript of 1431470 Bacterial Trans TG InHousePrinting · expression • To explore the concept of phenotype...