Bacterial Transformation Using Fluorescent Proteineds198hs.weebly.com/uploads/7/8/5/4/7854190/sciencebridge... · the Bacterial Transformation Using Fluorescent Protein lab: Cell

Bacterial TransformationUsing Fluorescent Protein

Teacher Guide

sciencebridge

ScienceBridge/UC San Diego

© 2011

All rights reserved.

Content written/prepared by the following:

UCSD - ScienceBridge

Jeremy Babendure

Alegra Bartzat

Maarten Chrispeels

Heather Gastil

Shelley Glenn Lee

Heather Liwanag

Johnnie Lyman

James Short

Cover Image: Transformed E. coli fluorescing under UV light. Blue, green, and grape fluorescent proteins are represented here (ScienceBridge PM1 mix).

TABLEOFCONTENTS

TableofContents

1 Program and Lab Overview 5

ScienceBridge Program . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Lab Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

2 Biology Curriculum 7

Lab Goals and Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . 7

CA State Standards Addressed . . . . . . . . . . . . . . . . . . . . . . . . 7

Content Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Research Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Implementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

PowerPoint Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Teaching Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Student Leader Preparation . . . . . . . . . . . . . . . . . . . . . . . 17

Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Assessment Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Teacher Key . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

P1 Protocol 1 21

P2 Protocol 2 27

A1 Appendix 1: Ecology of Fluorescent Proteins 35

PROGRAMANDLABOVERVIEW| 5

1 ProgramandLabOverview

ScienceBridge Program

About ScienceBridge

ScienceBridge is a Science Outreach Initiative based at the University of California, San Diego (UCSD) that serves secondary school teachers and students by connecting students to current and relevant scientific research through classroom activities, university experiences and community events. The foundation of the ScienceBridge program is our Teacher Profes-sional Development program, from which the following activity was developed at UCSD in collaboration with local science teachers and is now offered as a training and implementation package for the high school classroom.

One primary goal of ScienceBridge is to create very affordable and accessible labs that engage students with authentic science experiences. We work to optimize each activity to minimize the dependency on expensive equipment and other resources sometimes lacking at a school site. In doing so, we have created activities that can be implemented in virtually ANY class-room, but are also able to be “ramped up” or have added complexity to challenge more advanced students or to utilize avail-able classroom resources. ScienceBridge also supports and is helping to optimize student-run biotechnology sites within specific school districts that will allow materials to be available and sustainable over time, eliminating dependency on external resources.

Professional development & curriculaScienceBridge’s Teacher Professional Development strives to create connections between teachers and scientists, increase teachers’ and students’ access to current scientific information and resources, and encourage the engagement of students as leaders in the classroom. Each ScienceBridge teacher is trained to use the materials and lab protocol created at UCSD and brings a handful of students from his or her science classroom. These student leaders will learn to use the resources and serve as teaching assistants and resident “experts” in the classroom during activity implementation. All student and teacher input is encouraged and considered at all times, such that our training sessions, curriculum, and resources are the most effective and useful to the audience.

We are very pleased to offer these resources to you and hope you have a great experience with this lab activity!

For more information and program updates, visit:

http://sciencebridge.ucsd.edu

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Lab DescriptionBacterial transformation using fluorescent proteinsBacteria have the unique ability to acquire and express new traits by incorporating foreign DNA from the environment into their cells through their cell membranes. This process is called transformation and scientists utilize this process to create and study DNA, genes, and gene products such as proteins. In this ScienceBridge lab activity, students will transform wild-type E. coli bacteria with engineered DNA encoded with a gene for a fluorescent protein. In other words, normal bacteria will be given the ability to create glowing proteins, resulting in glowing, fluorescent bacteria! The fluorescent proteins originate from gene sequences that were optimized in the lab of Nobel Prize winner Dr. Roger Tsien at UCSD, and are widely used in scien-tific research to “tag” proteins of interest inside living cells in order to visualize cellular processes. What sets the ScienceBridge activity apart from other transformation activities is the rainbow of colors. Instead of just one glowing color, students see an array of colors that result from just slight mutations to the original fluorescent protein gene.

After completing this lab activity, students will have a better understanding of key biological and chemical processes such as transcription and translation, the nature of cell membranes, and protein structure and function. Students will also gain im-portant laboratory and technical skills while engaging in experimental design, data collection and analysis, communication of findings, and sources of experimental error. Students and teachers alike will benefit from learning about and utilizing cutting-edge fluorescent protein technology in their very own classroom.

The ScienceBridge Bacterial Transformation activity can be used in many ways within the biology curriculum (Genetics: Mutations, Ecology: Biodiversity, Evolution, Biotechnology) but may also benefit a Chemistry or Environmental Biology classroom. Additionally, the activity can be expanded by utilizing the ScienceBridge Protein Purification protocol, in which students learn how and why proteins are isolated, studied, and synthesized by scientists, and how these technologies are in-strumental to scientific progress.

BACTERIALTRANSFORMATIONUSINGFLUORESCENTPROTEIN

Greenfluorescentprotein.Notethebarrelshapeoftheprotein,withthechromophoreinthecenter.Theimageatrightshowsacutawaysothechromophorecanbeseenmoreclearly.

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2 BiologyCurriculum

Lab Goals and Objectives

In this lab, students will insert a gene that codes for a fluo-rescent protein into bacteria, changing the genotype. After the bacteria reproduce, transcribe, and translate the gene, stu-dents will observe the fluorescent color of the bacteria. This change in phenotype (fluorescence) is due to the fluorescent proteins inside the bacterial cells.

Lab ObjectivesAfter completing this activity students will be able to:

1. Understand the concept of an experimental control.

2. Define, identify, and explain the process of bacterial transformation.

3. Understand the central dogma of molecular biology (DNA --> RNA --> protein --> trait).

4. Explain how a change in genotype leads to a change in phenotype.

CA State Standards AddressedThe following CA state science standards are addressed in the Bacterial Transformation Using Fluorescent Protein lab:

Cell biologyThe fundamental life processes of plants and animals depend on a variety of chemical reactions that occur in specialized areas of the organism’s cells.

• Students know the central dogma of molecular biology outlines the flow of information from transcription of ribonucleic acid (RNA) in the nucleus to translation of proteins on ribosomes in the cytoplasm.

GeneticsGenes are a set of instructions encoded in the DNA se-quence of each organism that specify the sequence of amino acids in proteins characteristic of that organism.

• Students know the general pathway by which ribosomes synthesize proteins, using tRNAs to translate genetic information in mRNA.

• Students know how to apply the genetic coding rules to predict the sequence of amino acids from a sequence of codons in RNA.

• Students know how mutations in the DNA sequence of a gene may or may not affect the expression of the gene or the sequence of amino acids in an encoded protein.

The genetic composition of cells can be altered by incorpora-tion of exogenous DNA into the cells. As a basis for under-standing this concept:

• Students know the general structures and functions of DNA, RNA, and protein.

• Students know how genetic engineering (biotechnology) is used to produce novel biomedical and agricultural products.

• Students know how basic DNA technology (restriction digestion by endonucleases, gel electrophoresis, ligation, and transformation) is used to construct recombinant DNA molecules.

• Students know how exogenous DNA can be inserted into bacterial cells to alter their genetic makeup and sup-port expression of new protein products.

BIOLOGYCURRICULUM

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Investigation and ExperimentationScientific progress is made by asking meaningful questions and conducting careful investigations. As a basis for understanding this concept and addressing the content in the other four strands, students should develop their own questions and perform investigations. Students will:

• Select and use appropriate tools and technology (such as computer-linked probes, spreadsheets, and graphing calculators) to perform tests, collect data, analyze relationships, and display data.

• Identify and communicate sources for unavoidable experimental error.

• Identify possible reasons for inconsistent results, such as sources of error or uncontrolled conditions.

• Formulate explanations by using logic and evidence.

• Recognize the issues of statistical variability and the need for controlled tests.

Content InformationIntroductionTransformation is a simple yet powerful technology used by scientists to alter the genetic code of a living organism. By under-standing the central dogma of molecular biology and other biological and chemical processes, scientists have been able to take genetic code from one organism and give it to another, resulting in major advances in health, medicine, and agriculture. In this lab activity, students will alter the genetic makeup of bacterial cells by introducing a gene to produce a glowing protein.


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The central dogma of molecular biologyProteins are the building blocks of living organisms, performing specific functions within the body such as providing structure and support, carrying out enzymatic functions, regulating hormonal activities, and other vital processes. Proteins are coded by an organism’s DNA, specifically sequences within the DNA called genes. Every protein is produced through a series of pro-cesses called transcription and translation, in which a gene within a strand of DNA is “read” and transcribed into a strand of RNA, which is then “read” and translated into a protein using a collection of amino acids. This is known as the central dogma of molecular biology (DNA --> RNA --> protein --> trait). The central dogma applies to all living cells, from the smallest bacterium to the largest animal.

ThemolecularstructureofDNA,showingthespecificpairingofthenitrogenousbases(adenine:thymine,guanine:cytosine).

AcomparisonofthestructureofRNAandDNA.DNAiscomprisedofadouble-strandedhelixbasedonthesugardeoxyribose,and

utilizesthebasescytosine,guanine,adenine,andthymine.RNAiscomprisedofsingle-strandedhelixbasedonthesugarribose,and

utilizesthebasescytosine,guanine,adenine,anduracil.

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BacteriaBacteria are single-celled organisms classified as prokaryotes. They do not have nuclei, but they do have DNA. This DNA is found on a single, circular chromosome that contains all of the genes the bacterium needs for its normal existence (its genome). In addition, bacteria naturally contain one or more significantly smaller circular pieces of DNA called plasmids. Plasmid DNA contains genes for traits that may be beneficial to bacterial survival under certain environmental conditions. In nature, bacteria can transfer plasmids back and forth, allowing them to share these beneficial genes. This mechanism allows bacteria to adapt to new environments. The recent occurrence of bacterial resistance to antibiotics is due to the natural trans-mission of plasmids.

DNA can be exchanged between bacterial cells in three ways.

1. Conjugation (bacterial “sex”) involves the exchange of DNA through direct cell-to-cell contact or through a bridge-like connection between two cells called a sex pilus.

2. Transduction involves the transfer of DNA from one bacterial cell to another through a virus.

3. Transformation is the uptake of DNA from the environment surrounding the cell.

RESEARCHAPPLICATIONS

This unique ability of bacteria to move foreign plasmid DNA into their cells is utilized by scientists to produce and study a variety of proteins, including human proteins. Genes from one organism (e.g. human) can be cut from the original DNA strand and inserted into plasmid DNA, which may result in the bacteria producing the protein of interest (e.g. insulin).

Bacteria reproduce rapidly and are visible as colonies on a growth plate. Each colony on the plate is the offspring of one original bacterial cell (a clone of the original). A colony may represent millions of cells, all of which are genetically identical, since they all came from the same original bacte-rium. The bacterium replicates not only its own circular chromosome, but also its plasmid DNA. The bacteria utilized in this lab are Escherichia coli (E. coli), which are rod-shaped bacteria that are often found in the human digestive tract. E. coli are commonly used as model organisms in scientific research.

Genetic engineering

Scientists create plasmid DNA “vectors” to transfer genetic information between organisms. Scientists can determine the unique sequence of a gene, copy or change it, and insert it into another organism’s DNA through genetic engineering. When done in bacterial cells, this process is called transformation; when transformation is successful, a bacterium will be able to make proteins it would normally be unable to, possibly giving it traits it did not previously have. When scientists apply this technique to a multi-cellular organism, such as a plant or a mouse, and successfully alter its genetic makeup, it is called trans-genic transformation.

To conduct a transformation, the gene to be transferred is placed into a plasmid DNA vector. This is done with the help of restriction enzymes, which are naturally occurring enzymes from bacteria that recognize a particular sequence of DNA bases and cut the DNA at that sequence. Bacteria use restriction enzymes to protect themselves from viruses that inject their DNA into the bacteria; the enzymes can cut the viral DNA before it can hurt the bacteria. The same restriction enzyme is used to cut the ends of the gene to be transferred and to cut open the circular plasmid DNA vector. Because the cuts are made using the same restriction enzymes, the cuts have the same base sequence at the ends. These matching ends will match and reattach when placed together with the aid of another enzyme, DNA ligase. Plasmid DNA vectors containing fluorescent protein (and antibiotic resistance genes) have been constructed for use in this transformation lab.

Genetic transformation is used every day in many areas of biotechnology. In agriculture, genes coding for traits such as drought resistance can be genetically transformed into plants. In bio-remediation, bacteria can be genetically transformed with genes enabling them to digest hydrocarbons, to clean oil spills. Medical applications of transformation include the creation of proteins, such as insulin (synthesized by Genentech) and factor VIII (blood clotting protein synthesized by Bayer).

E. colicoloniesonagar

10 | BACTERIALTRANSFORMATIONUSINGFLUORESCENTPROTEIN

Bacterial Transformation

A bacterial cell has a cell wall and plasma membrane, which help the cell maintain an internal environment that is chemi-cally distinct from the external environment. The cell wall and cell membrane serve as barriers that prevent the passage of foreign material (including external DNA) into the cell. Because of these barriers, transformation can only occur under special conditions. In the laboratory, scientists use a combination of techniques to help move the plasmid DNA vector through the bacterial cell membrane.

In this lab, the bacteria are placed into a solution of calcium chloride (CaCl2). In solution, CaCl2 separates into calcium (Ca2+) and chloride (Cl-) ions. It is thought that the calcium ions help to “shield” the negatively charged phosphates on the DNA, helping the DNA pass through the phospholipids of the plasma membrane. The use of CaCl2 solution is combined with a procedure known as heat shock. The heat shock procedure involves placing the bacterial solution on an ice bath, then heat-ing the bacterial solution at a precise temperature for a very short period, and returning the bacterial solution to the ice bath. This procedure is thought to “loosen” the phospholipids and create spaces between them to allow the DNA to pass through the membrane. Placing the solution on ice slows the movement of the phospholipids and causes them to move close together. The heat shock temporarily causes the phospholipids to move very quickly, and this sudden movement makes the membrane

Transformedbacteriaunderplainlight TransformedbacteriaunderUVlight

AmpR

FP gene

Ampicillin resistance gene

more permeable to molecules like the DNA. Placing the bacterial solution on the ice bath after the heat shock causes the phospholipids to slow down and helps “seal” the new DNA inside the cell. Note that the heat shock procedure must be carried out very precisely: if the temperature is too hot or the time on heat is too long, the bacteria can die; if the tempera-ture is too cold or the time on heat is too short, the DNA may not have the chance to move across the membrane into the cell.

To select only the bacteria that have successfully been transformed with the fluorescent protein gene (in this lab), the plasmid DNA vector has also been engineered with a gene for resistance to an antibiotic known as ampicillin (Amp). Ampicillin inhibits the growth of the bacterial cell wall, which prevents the bacteria from growing. The ampicillin resis-tance gene codes for the production of the protein beta-lactamase, an enzyme that allows the bacteria to digest the antibiotic before it can cause any harm. If ampicillin is mixed into the agar (growth medium) on the bacterial plates, the only bacteria that can survive on these plates will be bacteria with the gene/plasmid for ampicillin resistance. Therefore, in the transformation procedure, if bacteria grow on the LB plates containing ampicillin, they must have been successfully transformed. Any one of the different colored fluorescent proteins can be inserted into the same place in this plasmid. In this lab, each DNA plasmid

Genes can be cut out of human, animal, or plant DNA and placed inside bacteria. Bacteria will then produce the “foreign” protein coded by the gene in large quantities for therapeutic treatment. For example, a healthy human gene for the hormone insulin can be put into bacteria and, under the right conditions, these bacteria can make authentic human insulin just as they would make their own proteins. This insulin protein is then purified from the bacteria (see Protein Purification lab), and used to treat patients with the genetic disease diabetes, in which the insulin gene does not function properly.

SchematicoftheplasmidusedintheSci-enceBridgetransformationlab.Theplasmidcontainsthegeneforafluorescentproteinandthegeneforresistancetoampicillin.

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vector has the gene that codes for one of six different fluorescent proteins. Plasmid mix 1 (PM1) has three different types of plasmids: one with the gene for green fluorescent protein (GFP), one with the gene for blue fluorescent protein (BFP), and one with the gene for Grape (a purple fluorescent protein). Plasmid mix 2 (PM2) is a mix of three more plasmids: one with the gene for Cherry (a purplish-pink protein), one with the gene for Tangerine (a bright pink protein), and one with the gene for yellow fluorescent protein (YFP). Your successfully transformed bacteria will fluoresce when exposed to a blacklight or UV light (see previous page).

FluorescenceFluorescence occurs when light of one wavelength (or color) is absorbed and a light at a different wavelength (or color) is re-emitted, usually at a slightly lower energy. Fluorescence can occur at any wavelength, but humans cannot observe much of the electromagnetic spectrum visibly and need special instruments to detect fluorescence at other wavelengths outside the visible spectrum. When utilizing fluorescence for research purposes, UV light is often the preferred wavelength for absorption because the lower energy wavelength emitted is typically in the visible spectrum. The fluorescent proteins used in this lab can glow because they contain a chromophore, a functional group which changes the shape of the molecule when excited by light. Note that the grape fluorescent protein in the ScienceBridge lab does not fluoresce under UV light. A higher energy source would be needed to cause fluorescence in the grape protein.

Research ApplicationsWith the advent of genetic engineering in the late 20th century, many researchers have been able to place the gene that makes their protein of interest into a plasmid. This allows them to make high quantities of the protein so that it can be studied biochemically in isolation from other macromolecules in a cell. For example, if one wanted to study the characteristics of an enzyme that causes an apple to turn brown after being cut, then he or she could isolate the gene for that enzyme, make a large quantity of the protein, and then test how changes in temperature or pH affect it in a test tube. Once understood in isola-tion, that information could be used to devise a way to alter the enzyme’s function within the apple. (See the ScienceBridge Enzyme/Substrate Reactions lab)

Isolating macromolecules and studying them separately from others has been a highly successful strategy to understand biological processes; however, some questions cannot be answered in this way. Sometimes studying the macromolecule while it is within the whole cell or organism would help a researcher answer a question best. But how do you see into a world that is beyond your eyes’ ability to perceive? Fluorescent proteins have been utilized much like placing a flashlight at the end of a protein. With the proper microscope, a protein that has been “tagged” with fluorescence can be observed within the cell, moving around and interacting with other macromolecules. Several labs at UCSD utilize this technology to increase our un-derstanding of cellular processes. The following labs utilize fluorescent proteins to see a new and wonderfully complex world within living cells.

Klemke LabDr. Richard Klemke, Pathology Department and Moores Cancer Center, UCSD

Utilizing a unique vertebrate model, the zebrafish, Dr. Klemke and his research team study how cancer cells invade other tissue in a process called metastasis. A cancer tumor needs a supply of nutrients and gas exchange to allow it to grow and gain access to the blood system, so that individual cells may move from the origi-nal site and grow into a secondary tumor. The Klemke lab utilizes high resolution microscopy to image zebrafish that have had their blood vessels tagged with GFP (green fluorescent protein). Two types of human cancer cells are injected into the fish. One type of human cancer is not metastatic and the other is metastatic; both are identified by a different color – blue and red, respectively. Therefore, the meta-static cells that attract new blood vessel growth can be studied using microscopy that allows for the creation of three-dimensional images of green blood vessels and red tumor cells.

http://klemkelab.ucsd.edu

Transgeniczebrafishwithbloodvesselsingreenwithtumorcellsinred.

IMPLEMENTATION


Jin LabDr. Yishi Jin, Division of Biological Sciences, UCSD

Understanding the development of nervous systems is the general area of study for the Jin laboratory members. Their laboratory website lists four questions that frame the studies conducted in the lab:

1. How are neurons that possess specific properties generated?

2. How are neurons guided to their targets?

3. How do neurons form synapses?

4. How do the synaptic connections remodel?

The answers to these questions are important because each neuron can form more than 1000 connections (synapses); how-ever, these connections are dynamic. When we learn something new or form memories, we are actively improving or degrad-ing connections between neurons. The model organism that the Jin lab uses is a transparent nematode approximately 1mm in length that is naturally found in soil. Its transparency allows researchers to see within the organism in all stages of its life (from fertilized egg to adult). By fluorescently tagging a protein involved in synapse development, scientists can study the protein’s actions within the whole organism at any developmental stage. Understanding the molecular basis of the processes involved could lead to significant advances in repairing spinal cord injuries.

http://biology.ucsd.edu/labs/yishijin/

Tsien LabDr. Roger Tsien, Skaggs School of Pharmacy and Pharmaceutical Sciences

Sharing the Nobel Prize in Chemistry with Dr. Osamu Oshimomura and Dr. Martin Chalfie, Dr. Roger Tsien helped develop the multiple fluorescent protein colors you see in the following image from GFP (green fluorescent protein) and RFP (red fluorescent protein). When the original GFP was made available to researchers to light up their fa-vorite protein to study, many researchers wanted to visualize their protein within a living cell or organism. With the development of the multiple colors, researchers could watch many proteins interact in real time. Fluorescent microscopes and computer software allowed for more detailed discoveries and even protein-to-protein interactions could be identified when two different fluorescent proteins would combine to produce a distinct color.

http://tsienlab.ucsd.edu/

Agarplateoffluorescentbacterialcolonies

GFPexpressioninC. elegans

Avarietyofnewandoldlocalizationmethodsareusedtovisualizecomponentsofaculturedhumanadenocarcinoma(HeLa)cell.Thenucleusislabeled

withasmall-moleculedye(blue),theGolgiapparatusisimmunolabeledwithquantumdots(yellow),micro-tubulesaregeneticallytaggedwithafluorescentpro-

tein(green),andtheactincytoskeletonislabeledwithatetracysteine/biarsenicalpair(red).Image:NationalCenterforMicroscopyandImagingResearch/B.N.G.

Giepmans.April14,2006

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Classroom ImplementationTeacher preparationOne kit will have enough materials for a maximum of one class of 40 students, though it is recommended that you use one kit per 32 students (eight groups of four students) so that you have extra materials. You can order enough kits for each of your classes to implement the lab activity. You will have three things to prepare for the lab at least the day before you implement.

Bacterial Transformation Kit ChecklistPlease contact your Tech Site immediately if you find that any items are missing or damaged:

Tech SiteSara Dozier ([email protected])

IMPLEMENTATION

Store at room temperature___ (40) Transfer pipettes

___ (10) Sterile transfer loops

___ (30) 2 mL clear round bottom microcentrifuge tubes

___ (20) Cotton swabs

___ (10) CaCl2 blue 1.5 mL tubes with 1 mL each [50mM]

___ (1) TE Buffer orange 1.5 mL tube with 1 mL total (10mM Tris, 1mM EDTA)

___ (10) Micropipette tips (for pre-streaking class plates)

___ (1) Bacterial Stab (for making pre-streaks) (Note: one stab/teacher - not one stab/kit)

Store at 4°C (refrigerated) Agar Plates

___ (20) Small LB/amp agar plates (with red line)

___ (20) Small LB only agar plates (no line)

Plasmid Mix Bag

___ (1) PM1 green 0.6 mL tube: 15 μL @ 165ng/μL

___ (1) PM2 purple 0.6 mL tube: 15 μL @ 165ng/μL

Items not included in the kit___ (1) Sharpie marker

___ (1) Cup with ice - crushed ice or ice/water mix

___ (1) Waste container

___ (1) Hot water bath at 42°C

UCSD Lab materials and Curriculum: Heather Gastil ([email protected])

Prior to implementation___ Print protocols

___ Aliqout DNA for lab groups

___ Make pre-streak bacteria plates (1-3 days before lab)___ Prepare lab materials for each station

Day Before Implementation (3 days if not using incubator)(Do not leave “pre-streak” plates in incubator for more than 24 hrs)

___ Prepare bacteria starter plates or “pre-streaks”

___ Aliquot DNA plasmids for lab groups

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Prepare bacteria “pre-streak” platesMaterials needed:

___ (10) LB only plates (no line)

___ Micropipette tips (found in bag of dry materials)

___ Bacterial stab (store at room temperature)

Note: The plates can be left at each lab station throughout the day.


1. Using a permanent marker, label the bacterial plate on the bottom of the plate, along the edges, with “pre-streak” and the date.

2. Open the stab, and poke a sterile pipette tip down into the stab 3-4 times. Close the stab.

3. Gently smear the bacteria on one edge of the LB agar plate (see diagram at right).

4. Discard the pipette tip in a biohazardous waste container*.

5. Using a new, sterile pipette tip, gently streak 4-5 lines from your original smear on the LB agar plate.


7. Using a new, sterile pipette tip, gently streak 4-5 lines from the last set of lines, being careful to avoid the original smear.


9. Using a new, sterile pipette tip, gently streak 4-5 lines from last set of lines, being careful to avoid the original smear.


11. Incubate the pre-streak either at 37ºC overnight or 2-3 days if left at room temperature.

Note: Do not store plates in the refrigerator - only store plates at room temperature.

* Any materials that have touched bacteria are considered biohazardous waste. For safety compliance, you should collect these materials in a rigid container and sterilize them by soaking them in a 10% bleach solution prior to discarding them. Once sterilized, the materials may be discarded with the regular trash.

Aliquoting DNA Plasmids for lab groupsMaterials Needed:

___ (1) PM1 tube (green)

___ (1) PM2 tube (purple)

___ (10) Clear 2 mL round bottom microcentrifuge tubes

___ (1) Orange 1.5 mL microtube TE buffer (1.0 mL of TE)

1. Add 0.5 mL of TE buffer to each PM1 and PM2 tube. This will almost fill up the PM1 and PM2 tubes. Gently pipette up and down and then firmly close the tubes and invert them several times to ensure mixing. Make sure to change pipette tips between mixes, so as not to transfer plasmids between tubes.

2. Using the provided clear 2 mL round bottom microcentrifuge tubes, label the caps: (5) tubes PM1 and (5) tubes PM2.

3. Aliquot 0.1 mL into your labeled PM1 and PM2 tubes. You should have a total of (10) aliquots - 5 PM1 and 5 PM2. Again, make sure to change pipette tips between plasmid mixes to avoid transferring extra plasmids.

Note: The plasmid DNA in both PM1 and PM2 is concentrated (165 ng/μL), so there is only 15 μL present in the tube before diluting. This is a small amount, so check the tubes for the 15μL before diluting. Often, some of the liquid gets caught in the cap of the tube. It helps to spin the tubes briefly in a centrifuge, to bring the liquid down into the bottom of the tubes.

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Group materials___ (1) Bacterial pre-streak (with bacterial colonies)

___ (2) LB/Amp plates (red line=ampicillin)

___ (1) LB only plate (no red line)

___ (2) 2.0 mL round bottom microcentrifuge tubes

___ (4) Disposable transfer pipets

___ (1) Sterile inoculation loop


Place the following on ice___ (1) 0.1 mL plasmid aliquot (PM1 or PM2)

___ (1) Blue 1.5 mL tube containing CaCl2

IMPLEMENTATION

Items not included in kit___ (1) Sharpie marker

___ (1) Cup with ice - crushed ice or ice/water mix


___ (1) Hot water bath at 42°C

FOR CLASS WASTE:

Prepare a 10% bleach solution in a tub or sink to soak all waste materials. Soak materials (pipettes, plates, etc.) for about 20 min in the solution and then they can be thrown in a regular trash.

Assembly of station materialsHave students label and assemble their group’s materials into a plastic bag. This acquaints them with the materials and helps expedite set-up.

Note: There should also be at least one hot water bath or heating block set at 42°C (no higher). A hot water bath can be made with a beaker of water and a hot plate.

GlossaryAmpicillin (Amp)An antibiotic (chemically similar to penicillin) that works by inhibiting the synthesis (building) of bacterial cell walls. Mixed with LB agar, it is used in this lab to select for trans-formed bacteria possessing the antibiotic resistance gene (the gene for beta-lactamase) on the plasmid. Those that do not have the plasmid with the antibiotic resistance gene (un-transformed bacteria) will die in the presence of ampicillin.

Bacterial StabA small vial containing the ScienceBridge E. coli strain, un-transformed, within LB agar. It is used for long-term storage of bacteria and can be kept at room temperature (for several weeks) or refrigerated (for a few months).

Bacterial ColonyA visible cluster of bacteria growing on an LB agar plate, cultured from a single bacterium. Each colony should contain only bacteria that are genetically identical. Even a small colony can contain hundreds of thousands of bacteria.

BacteriumThe singular form of bacteria. A single E. coli bacterium is rod-shaped, approximately two microns (μm) in length, and half a micron wide.

CaCl2Calcium chloride. During the transformation procedure, the Ca2+ ions, formed when CaCl2 is put into the solution theo-retically neutralize the negatively charged DNA, therefore facilitating the movement of the DNA from outside of the bacterial membrane to the inside of the cell. Also, the ions can shield the negative charges on the phospholipids of the cell membrane, once the movement of the phospholipids is slowed by placing the sample on ice.

Central DogmaThe central dogma of molecular biology. This idea, first put forth by Francis Crick in 1958, states that information in living cells flows from DNA to RNA to protein, and never in the reverse direction. This is the basis for a fundamental understanding of molecular biology.

Escherichia coliA gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of endothermic (warm-blooded) animals. Commonly used as a model organism and more commonly known as E. coli, this bacteria can also be found outside the human body among fecally contaminated envi-ronments. Most strains of E. coli, including the one used in this lab, are not harmful. The laboratory strain of E. coli has had its entire genome sequenced.


FluorescenceThe absorption of electromagnetic radiation (light) by an atom at a higher energy and the re-emission of electromag-netic radiation at a lower energy. See Appendix 1

GeneA stretch of DNA that codes for a particular product (usually a protein) that serves a function inside or outside the cell.

GenomeAll of an organism’s hereditary information (its genes).

GenotypeThe genetic makeup of an organism, usually in reference to a particular characteristic. In other words, the genotype refers to the specific “versions” of the genes in an organism’s DNA.

Heat ShockA procedure used during bacterial transformation to facili-tate the movement of DNA across the plasma membrane. In this procedure, the bacterial cells are kept on ice, then placed at 42°C for 45 seconds, and immediately put back on ice. Theoretically, heat shock helps to open spaces between the phospholipids of the plasma membrane, through which the DNA can pass into the cell.

LB AgarLB stands for lysogeny broth, which contains nutrients that support bacterial growth. While there is not one standard mix for LB, it generally contains peptides and casein pep-tones, vitamins, trace elements such as nitrogen and sulfur, and minerals. Agar is a gelatinous substance that most bacte-ria cannot digest.

Model Organism A non-human organism used in scientific research to under-stand key aspects of biology, with the anticipation that the results can help scientists understand the workings of other organisms as well.

PhenotypeA characteristic that results from the expression of a gene. A phenotype could manifest in the organism’s appearance (e.g. color) or some other function (e.g. antibiotic sensitivity or resistance).

PlasmidA small circular piece of DNA that replicates autonomously within the bacterium. It is referred to as extrachromosomal DNA because the bacterium has a single, circular chromo-some that contains all of its regular genes. Plasmids replicate themselves (separately from the bacterial chromosome), so bacteria usually have multiple copies of the same plasmid in a single cell.

PM1 (Plasmid Mix 1)The plasmid DNA mixture containing three different plas-mids with three different fluorescent protein genes: grape, blue, and green.

PM2 (Plasmid Mix 2)Plasmid DNA mixture containing three different plasmids with three different fluorescent protein genes: cherry, tanger-ine, and yellow.

TE BufferA buffer containing Tris base [10 mM], a common pH buf-fer, and EDTA [1mM], a molecule that chelates cations, that is used to dilute the plasmid DNA. It helps maintain the pH and preserves the DNA.

TranscriptionThe process by which RNA is synthesized (made) by copying a sequence of an organism’s DNA (a gene).

TransformationA process in which bacteria take up foreign DNA from the environment. Once that happens, the genes may be ex-pressed. Introduction of foreign DNA into eukaryotic cells is usually called transfection.

TranslationThe process by which a protein is synthesized (made). Dur-ing translation, messenger RNA (mRNA) is decoded by a ribosome, which coordinates the binding of transfer RNA (tRNA) to bring together amino acids in the correct se-quence.

| 17IMPLEMENTATION

PowerPoint NotesThe PowerPoint presentations that were given at both the teacher and student modules of the ScienceBridge training are available on our website for your use in the classroom.

Embedded in the notes section of the student PowerPoint presentation are talking points for the presentation that can printed out to use for your classroom lecture.

Download the files at http://sciencebridge.ucsd.edu. Once at the website, scroll over the top menu where it says Programs=>ScienceBridge Labs=>Content Areas=>Transformation

On the page you will see a link titled PowerPoints. Click on the button and download the student PowerPoint.

Teaching StrategiesWe suggest that you organize students in groups of four and have no more than eight lab groups. We give you enough materi-als for 10 groups, but using only eight groups is best to have extra materials on hand.

We have found that if you have the time, it helps to give the protocol to your students before the day of the implementation so they can read through the steps. Assigning roles to the students ahead of time also helps the lab run smoothly on the day of implementation.

It is also useful to print the Transformation Protocol Flow Chart (pg. 34 of this guide) for each lab station.

The curriculum is designed to be completed in three, 50-minute periods.

• Day 1 – Prepare the students with the Student PowerPoint and pre-lab material. Organize lab groups and prepare station materials.

• Day 2 – Lab protocol

• Day 3 – Results, analysis and conclusion

Student leader preparation• Meet with your student leaders before the imple-

mentation to answer any questions and review the lab procedure with them.

• Discuss how they can guide their fellow classmates rather than just taking over the step or telling them the answer.

• If you are not having your classes prepare their station materials, this a good task with which your student leaders can assist you.

• If you have student leaders assisting you in the classroom for implementation, we suggest assigning them to certain lab stations to guide the students at each station through the protocol.

ProtocolsThere are two versions of the Bacterial Transformation Using Fluorescent Protein protocol. All protocols contain pre- and post-lab questions to guide student learning.

• Protocol 1 – This protocol is text only with no pictures.

• Protocol 2 – This protocol is the same as the proto-col that you used in the training, and has pictures to assist your visual learners.

Assessment StrategiesThis lab includes two stages of assessment in the protocol. Within the lab protocol, a pre-lab question set is designed to assess student understanding of the Powerpoint material and prior knowledge of the lab subject area. A post-lab is provided for two reasons: (1) to assess student understanding of results from the lab and (2) to allow for a comparison between all groups’ data and promote sharing of data among groups. This will allow students to check their understanding of the results and to see all six fluorescent protein colors.

18 |

Bacterial Transformation Using Fluorescent Protein Teacher Key Student pre-lab questions1. What is a bacterial colony? How do the genotypes of individual bacteria in the same colony compare to each other?

A bacterial colony is a cluster of bacteria that originated from a single bacterial cell. All the bacteria in the same colony should be genetically identical, so their genotypes should be exactly the same.

2. What is a gene? What processes occur to make a protein from a gene?

A gene is a section of DNA that codes for a product (often a protein) that serves some function for the cell. To make a protein, the gene must first be transcribed into messenger RNA (mRNA) and the mRNA must be trans-lated into the protein.

3. What is a plasmid?

A plasmid is a small, circular piece of DNA that is separate from the main chromosome in a bacterial cell.

4. What is transformation?

Transformation is a process in which bacteria take up foreign DNA from the environment.

5. What do the “+” and “-” on the microtubes indicate about the contents of each tube in the transformation procedure?

The “+” indicates that the plasmid has been added to the tube, and the “-” indicates that no plasmid has been added.

6. What genes are present on the plasmid in this lab, and what is the function of each protein product?

The plasmid has a gene for a fluorescent protein, which makes the bacteria fluorescent (glows under UV light). The plasmid also has a gene for ampicillin resistance, which allows the bacteria to grow in the presence of the antibiotic ampicillin.

7. What does the red line on two of the plates indicate?

The red line means there is ampicillin in the plate.

8. What is the purpose of ampicillin (antibiotic) in the transformation procedure?

Ampicillin is used to select for the bacteria that have been transformed. Because the plasmid with the fluorescent protein gene also has a gene for ampicillin resistance, only the transformed bacteria can survive in the presence of ampicillin.


| 19TEACHERKEY

Plate # #1 LB/Amp (+)(red line) #2 LB/Amp(-)(red line) #3 LB/No Amp(-)(no red line)Plasmid present? YES NO YES NO YES NOAmpicillin present? YES NO YES NO YES NOExpected Plate Results (Drawing)

Students should draw a plate with many indi-vidual colonies.

Students should draw a plate with no growth.

A plate with continuous bacterial growth.

Expected Plate Results (Description)

Transformed colonies (col-ored bacteria)

No growth Bacterial “lawn”

Post-lab questions1. Estimate the number of fluorescent colonies that grew on your experimental plates.

Transformation efficiency varies. If students have too many colonies to count, they can estimate the total number of colonies by counting the colonies on one quadrant of the plate and multiplying by four.

2. Which plasmid mix did you have (PM1 or PM2) and which three colors of fluorescent colors of bacteria did you observe? (Yes, three colors are present! Look again if you did not see them all!)

PM1: Green fluorescent protein (GFP), Blue fluorescent protein (BFP), GrapePM2: Cherry, Tangerine, and Yellow fluorescent protein (YFP)Note: The bacteria expressing BFP will look like “normal” colonies under white light but glow blue under UV light. The Grape protein looks purple under white light and does not fluoresce under UV light (it requires a higher energy light than UV to fluoresce).

3. What made it possible for the colonies to be different colors?

Each colony came from a single transformed bacterial cell that took in one of the plasmids with a fluorescent pro-tein gene. There were three different kinds of plasmids in each mix (one for each color).

4. Describe how the plasmid was able to enter the cell. (Hint: There are two processes in the protocol that were designed to help the plasmid get into the cell. What are they?)

Placing the bacteria in a solution of calcium chloride helped to neutralize the DNA, because the calcium (Ca2+) ions “shielded” the negative charge on the DNA phosphates. Since charged molecules usually cannot pass through the hydrophobic barrier of the plasma membrane, neutralizing the charge helps the plasmid move into the cell.

20 |

The heat shock procedure also helps the plasmid enter the cell. During the heat shock, the phospholipids in the plasma membrane go from moving very slowly (when on ice) to moving very quickly (during the heat shock) and then moving slowly again (when back on ice). When the phospholipids suddenly move very quickly, spaces are created in the plasma membrane through which the plasmid can pass.

5. There are two controls in this experiment: the LB/Amp (-) plate and the LB/No Amp (-) plate. What are they testing? Assuming the lab procedure was performed correctly, what would it mean if bacteria grew on the LB/Amp (-) plate? What would it mean if no bacteria grew on the LB/No Amp (-) plate?

The LB/Amp (-) plate tests whether the ampicillin is working properly. The LB/Amp (-) plate contains ampicil-lin and the bacteria were not given the plasmid. If bacteria grow on the LB/Amp (-) plate, it could mean that the ampicillin was not working because the ampicillin should kill any bacteria that do not have the plasmid.The LB/No Amp (-) plate tests whether the bacteria are viable. The LB/No Amp (-) plate does not contain am-picillin and the bacteria were not given the plasmid. If no bacteria grow on the LB/No Amp (-) plate, it could mean that the bacterial cells were unable to grow. (If the heat shock procedure is too hot or too long, the bacterial cells can die.)

6. How can transformation be used in the medical industry or in research?

Because transformation allows bacteria to express a gene that has been engineered, it is widely used in medicine and research to produce proteins in bacterial cells. Many proteins (e.g. insulin, epinephrine) can be produced in bacterial cells. Scientists can also insert a gene into a cell to see what it does - they change the genotype to observe the effects on the phenotype and learn the purpose of the gene. Fluorescent proteins are produced by bacteria and used in research to “tag” other proteins or molecules and to visualize cellular processes.


| 21

Central questionHow does a change in the genotype of an organism affect its phenotype?

Overview of experimentIn this lab, you will insert a gene that codes for a fluorescent protein into bacteria, changing the genotype. After the bacteria reproduce, transcribe, and translate the gene, you will observe a change in the phenotype (appearance) of the bacteria.

How will the addition of a gene for a fluorescent protein affect the phenotype of the bacteria? − Hypothesis

Student pre-lab questions1. What is a bacterial colony? How do the genotypes of individual bacteria in the same colony compare to each other?





BacterialTransformationUsingFluorescentProteinPROTOCOL1

22 |







Based on your answers to the questions above, predict the results of your transformation procedure in the table below.

| 23PROTOCOL1

Materials checklist___ (1) ScienceBridge Transformation Protocol

___ (1) Bacterial pre-streak plate with bacterial colonies

___ (1) Agar plate containing LB/No Amp (no red line)

___ (2) Agar plates containing LB/Amp (red line)

___ (1) Sterile inoculating loop

___ (4) Plastic transfer pipettes

___ (2) Clear 2.0 mL microtubes

___ (2) Cotton Swabs

___ (1) Sharpie

___ (1) Piece of tape for sealing plates after inoculation


___ (1) Styrofoam cup with ice

ON ICE

___ (1) Blue 1.5 mL tube of CaCl2

___ (1) Clear 2.0 mL tube of plasmid DNA labeled either “PM1” or “PM2”

Shared materials for class

___ Hot water bath at 42ºC

Role in Group Student NameMaterialsReaderTimer

Technician

NOTE: When labeling plates, write on the BOTTOM of the plate (not the lid) and keep writing small and close to the edges!

1. Label plates all three plates with the date, class period and your group number or initials as listed below:

Plate # Label

1 “LB/Amp (+)” (positive)

2 “LB/Amp (–)” (negative)

3 “LB/No Amp (–)” (negative)

Lab Procedure

Group # Assign group roles:

Materials: Gathers and organizes group materials, and assists with disposal.

Reader: Reads the protocol aloud so group members can follow the steps.

Timer: Keeps track of timing for all timed steps. This is especially important for the heat shock!

Technician: Carries out the actual steps of the protocol.

LB/Amp (+) Name/Period

#1

LB/Amp (-) Name/Period

#2

LB/No Amp (-) Name/Period

#3

24 |

2. Close the caps on two microtubes and label each cap: one tube with a “+”, other tube with a “–”

3. Using a plastic transfer pipette transfer 0.5 mL of CaCl2 to each tube, close and place both tubes on ice for at least 2 minutes. Discard the pipette in the waste container.

4. Using a sterile loop, gently collect ONE colony (“dot”) of bacteria from the top of your bacterial starter plate, being care-ful not to gouge the agar. Transfer the collected colony to one tube of CaCl2. Swirl and twist the loop to make sure all the bacteria mix with the CaCl2 solution. Mix the contents by inverting the tube or flicking. The solution should look cloudy with no chunks.

5. Transfer ONE additional colony to the second tube by repeating instruction #4. Place the tubes back on ice.

6. Using a clean plastic pipette, add all of the plasmid mix solution (labeled either “PM1” or “PM2”) into the positive (+) tube with CaCl2. BE SURE THAT THE PLASMID IS ONLY TRANSFERRED TO THE + TUBE. Mix. Discard the used pipette.

7. Incubate both tubes on ice for 10 minutes. Make sure the tubes are immersed in the ice.

8. The timer and one other group member will:

• Check the water bath temperature to ensure it is at 42ºC.

• Hold the tubes in the hot water for exactly 45 seconds. Make sure that the tubes are in contact with the hot water.

• Immediately return the tubes to the ice for 2 minutes.

9. Invert your tubes gently to mix. Using a new pipette, transfer 0.25 mL of the cell mixture from the (-) negative tube to the LB/Amp (-) agar plate. Spread the mixture around the plate gently with a clean cotton swab.

10. With the same pipette, transfer 0.25 mL of the cell mixture from the (-) negative tube to the LB/No Amp (-) agar plate. Spread the mixture around the plate gently with the same cotton swab. Make sure to completely finish the two “-“ (nega-tive) plates before spreading the cells on the “+” (positive) plate.

11. With a new clean pipette, transfer 0.25 mL of the cell mixture from the (+) positive tube to the LB/Amp (+) plate. Spread the mixture around the plate gently with a clean cotton swab.

12. Stack your plates, tape them together, put plates UPSIDE DOWN to grow overnight in 37ºC incubator.


| 25PROTOCOL1

Now that you have performed a transformation, fill out the table below to describe your results.

Plate # #1 LB/Amp (+)(red line) #2 LB/Amp(-)(red line) #3 LB/No Amp(-)(no red line)Plasmid present? YES NO YES NO YES NOAmpicillin present? YES NO YES NO YES NOActual Plate Results (Drawing)

Actual Plate Results (Description)

1. Estimate the number of fluorescent colonies that grew on your experimental plates.



Post lab questions

26 |




Conclusion / summary (revisit hypothesis)


| 27PROTOCOL2

Central questionHow does a change in the genotype of an organism affect its phenotype?

Overview of experimentIn this lab, you will insert a gene that codes for a fluorescent protein into bacteria, changing the genotype. After the bacteria reproduce, transcribe, and translate the gene, you will observe a change in the phenotype (appearance) of the bacteria.

How will the addition of a gene for a fluorescent protein affect the phenotype of the bacteria? − Hypothesis

Student pre-lab questions1. What is a bacterial colony? How do the genotypes of individual bacteria in the same colony compare to each other?





BacterialTransformationusingFluorescentProtein







Based on your answers to the questions above, predict the results of your transformation procedure in the table below.

| 29PROTOCOL2

Materials checklist___ (1) ScienceBridge Transformation Protocol

___ (1) Bacterial pre-streak plate with bacterial colonies

___ (1) Agar plate containing LB/No Amp (no red line)

___ (2) Agar plates containing LB/Amp (red line)

___ (1) Sterile inoculating loop

___ (4) Plastic transfer pipettes

___ (2) Clear 2.0 mL microtubes


___ (1) Sharpie

___ (1) Piece of tape for sealing plates after inoculation


___ (1) Styrofoam cup with ice

ON ICE

___ (1) Blue 1.5 mL tube of CaCl2

___ (1) Clear 2.0 mL tube of plasmid DNA labeled either “PM1” or “PM2”

Shared materials for class

___ Hot water bath at 42ºC

Role in Group Student NameMaterialsReaderTimer

Technician

BacterialTransformation

Lab procedure NOTE: When labeling plates, write on the BOTTOM of the plate (not the lid) and keep writing small and close to the edges!

1. Label plates all three plates with the plate number, date, class period and your group number or initials as listed below:

Plate # Label

1 LB/Amp (+) (red line)

2 LB/Amp (–) (red line)

3 LB/No Amp (–) (no red line)

LB/Amp (+) Name/Period

#1

LB/Amp (-) Name/Period

#2


#3

Group #

Assign group roles:

Materials: Gathers and organizes group materials, and assists with disposal.

Reader: Reads the protocol aloud so group members can follow the steps.

Timer: Keeps track of timing for all timed steps. This is especially important for the heat shock!

Technician: Carries out the actual steps of the protocol.

30 |

3. Using a plastic transfer pipette transfer 0.5 ml of CaCl2 to both tubes, close and place them on ice for at least 2 minutes. Discard the pipette in the waste container.

+DNA(plasmid)

10 minutes

+ -

CaCl2 0.5 mL

4. Using a sterile loop, gently collect ONE colony (“dot”) of bacteria from the top of your bacterial pre-streak plate. Transfer the collected colony to one tube of CaCl2. Swirl and twist the loop to make sure all the bacte-ria mix with the CaCl2 solution. Mix the contents by inverting the tube or flicking the bottom of the tube. The solution should look cloudy with no chunks.

5. Transfer ONE additional colony to the second tube and repeat instruction #4. Place the tubes back on ice.

6. Using a clean plastic pipette, add all of the plasmid mix solution (labeled either “PM1” or “PM2”) into the positive (+) tube with CaCl2 BE SURE THAT THE PLASMID IS ONLY TRANSFERRED TO THE + TUBE. Mix. Discard the used pipette.

7. Incubate both tubes on ice for 10 minutes. Make sure the tubes are immersed in the ice.


+ -2. Close the caps on two microtubes and label each cap

with a sharpie: one tube with a “+”, other tube with a “–”

| 31PROTOCOL2

+

42o

37o

45 seconds 2 minutes

0.25 mL

0.25 mL

-

8. The timer and one other group member will

• Check the water bath temperature to ensure it is at 42ºC.

• Hold the tubes in the hot water for exactly 45 seconds. Make sure that the tubes are in con-tact with the hot water.

• Immediately return the tubes to the ice for 2 minutes.

9. Invert your tubes gently to mix. Using a new pipette, transfer 0.25 mL of the cell mixture from the (-) nega-tive tube to the LB/No Amp (-) agar plate. Spread the mixture around the plate gently with a clean cotton swab.

10. With the same pipette, transfer 0.25 mL of the cell mix-ture from the (-) negative tube to the LB/Amp (-) agar plate. Spread the mixture around the plate gently with the same cotton swab. Make sure to completely finish the two “-” (negative) plates before spreading the cells on the “+” (positive) plate.

11. With a new clean pipette, transfer 0.25 mL of the cell mixture from the (+) positive tube to the LB/Amp (+) plate. Spread the mixture around the plate gently with a clean cotton swab.

12. Stack your plates, tape them together, put plates UPSIDE DOWN to grow overnight in 37ºC incubator.

0.25 mL

-

Plate #3

LB/No Amp (-)

Plate #2

LB/Amp (-)

Plate #1

LB/Amp (+)


#3


Now that you have performed a transformation, fill out the table below to describe your results.

Plate # #1 LB/Amp (+)(red line) #2 LB/Amp(-)(red line) #3 LB/No Amp(-)(no red line)Plasmid present? YES NO YES NO YES NOAmpicillin present? YES NO YES NO YES NOActual Plate Results (Drawing)

Actual Plate Results (Description)

1. Estimate the number of fluorescent colonies that grew on your experimental plates.



Post-Lab Questions

| 33PROTOCOL2




Conclusion / summary (revisit hypothesis)

34 |

Transformation Protocol Flow Chart

Positive tube (+) Negative Tube (-)

500 μL CaCl2ON ICE!

500 μL CaCl2ON ICE!

One (1) Bacteria Colony One (1) Bacteria ColonyVortex or Shake to Mix Vortex or Shake to Mix

100 μL Plasmid (PM1 or PM2) No Plasmid Tap Tube Gently

ICE HEAT ICE ICE HEAT ICE45 Second Heat Shock 45 Second Heat Shock

100 μL on LB/Amp (+) Plate 100 μL on LB/Amp (-) and

LB Only (-) Plates

LB/Amp (+) LB/Amp (-) LB Only (-)

+ -

AmpR

FP gene

Ampicillin resistance gene


| 35

The discovery of fluorescent proteins has been revolutionary in its applications for medical and biological research (bio-technology). But where did the original proteins come from? Why do they exist? How do they work in nature? And what organisms use fluorescence and why?

The many-colored fluorescent proteins used in the transfor-mation activity all originated from one of two proteins found in nature: the green fluorescent protein (GFP)— discovered in and isolated from a marine jellyfish (Aequorea victoria) and red fluorescent protein (dsRed), a protein more recently discovered in a particular group of corals (“mushroom” corals, Discosoma spp.). These fluorescent proteins are light harness-ing and light emitting molecules whose function in nature remains a mystery. Meanwhile, fluorescent proteins have been and continue to be extensively studied and engineered in the research lab as powerful molecular markers.

While studying the bioluminescence of the crystal jelly, Shimomura found it odd that the light created by the jelly was blue, yet the color being expressed by the jelly was green! How and why does this jelly convert its blue light to green? The answer to “how” is that the jelly was producing a protein that absorbed the blue light and emitted green light. Shi-momura discovered this protein and named it “GFP” (green fluorescent protein). He described its structure and func-tion which provided the foundation for decades of scientific progress in molecular research. However, no scientist has yet figured out why the jelly prefers green light to blue, or how the jelly uses its light in the ocean.

Fluorescence vs . bioluminescenceIn the deep sea, more than 90% of organisms are capable of producing their own light through bioluminescence. From bacteria to squid to fish, bioluminescence is an adaptation for survival and is used for either finding food by locating or attracting prey (e.g. lure of anglerfish, below), for protection against predators (e.g. counter-illumination in the hatchet-fish), or for communication between individuals of a given species (e.g. light patterns in firefly squid.)

Discovery of GFPIn the 1960’s, a marine biologist named Osamu Shimomura was interested in the bioluminescent behavior of the crystal jellyfish Aequorea victoria. Bioluminescence is the ability of a living organism to create its own light through a chemical reaction. In the sea, this adaptation (a behavior or structure that is passed from one generation to another and allows an organism to better survive within a particular environ-ment) is prominent in the sea, especially the deep sea, where fish, shrimp, and other organisms use light to attract mates, find food, or protect themselves from harm. The crystal jelly studied by Shimomura is not a deep sea organism, but is abundant in the waters of the Pacific Northwest.

Aequorea victoria

In most cases of bioluminescence, the light is created through a chemical reaction between a substrate (“luciferin”) and an enzyme (“luciferase”) in the presence of oxygen. The light is produced in an organ called the photophore and one or more photophores are strategically located on the body of an organism depending on how the light is used.

Appendix 1 - Ecology of Fluorescent Proteins

APPENDIX1

Femaleanglerfish

36 |

Although organisms are seemingly capable of producing dif-ferent colors of light, blue is the most common color used by marine organisms. Blue light (short wavelength light) tends to scatter in water and penetrate farther than colors such as red and orange (long wavelength light), which tend to be ab-sorbed by water rather quickly. If you take a red object under water with you, the deeper you go the less red it will appear. This happens because there is a decreasing amount of red light available to reflect back to your eye. (Remember that a red apple appears red because it absorbs all other colors and reflects red light. If there is no red light available, the apple will look gray or black). To take advantage of this phenom-enon, many deep sea organisms are red in color, as there is no red light available to reflect off of them, thus they “blend” into the darkness rather well. In the sea, the blue light cre-ated by bioluminescence is definitely meant to be seen!

If blue light is more effective than others when it comes to animal adaptations and the use of light in the sea, why are fluorescent proteins (FP’s) used to produce green, red, orange, and other colors of light?

Cnidarian biology and biodiversityThe many-colored fluorescent proteins used in this activity originated from jellyfish and corals, related organisms that belong to the phylum Cnidaria--stinging celled animals that are mostly found in the ocean and include sea anemones. In a research laboratory at UC San Diego, GFP (from jellyfish) and dsRed (from coral) were genetically altered (mutated) to create a library of proteins that would both absorb and emit different wavelengths (colors) of light under different environmental conditions. Scientists continue to research the existence, diversity and function of FPs in nature, especially as it relates to animal biology and ecology.

Research on the function of FPs in coral biology and health is currently being pursued by researchers at several universi-ties, including UCSD’s own Scripps Institution of Oceanog-raphy. Some hypotheses regarding the function of fluorescent proteins in corals include: they act as sunscreen, protecting the coral from the suns harmful rays; they act to convert the energy of the sunlight into light that can drive photosynthe-sis; they provide a beacon to coral symbionts or other coral-inhabiting microbes that can detect light.

To understand why scientists are studying these potential functions, you may want to know more about coral biology. The coral reef habitat is unique because it is warm, shallow, and crystal-clear (nutrient-poor) water. Corals and anemo-nes have a special symbiotic relationship (a relationship that is beneficial to both species) with unicellular algae called zooxanthellae. These organisms are dinoflagellates, a group of

microscopic plants which are usually found swimming and floating in the sea. Organisms that live like this are called plankton, and those that are plants are called phytoplankton. Like most plants, phytoplankton are able to convert the sun’s energy into food through a process called photosynthesis, so to survive they are only found in the upper layers of the sea and lakes where sunlight can penetrate.


Ahealthyreefenvironment.

PhotocourtesyofBirchAquariumatScripps

| 37

Appendix 2 - Structure determines function of fluorescent proteinsThe science within GFPGreen fluorescent protein (GFP) is a barrel-shaped protein with a unique abil-ity: when exposed to blue light, it can fluoresce green. The structure of GFP is quite elegant. Proteins are made up of amino acids that are linked together via an amide bond (N-C). There are 20 amino acids, and they are linked sequen-tially in different and distinct combinations to produce a long peptide polymer. Each amino acid has a different side chain, which has a certain chemistry to it that makes each amino acid different. GFP is made of 238 amino acids.

The 238 amino acids are linked together to form the basis for GFP. This se-quence is called the protein’s primary structure. There is a secondary structure, dictated by the chemistry of the protein’s amino acids’ amide bonds. Since proteins are hydrophilic, or water loving, the amino acids arrange themselves to hydrogen bond in the most energetically favorable state with water molecules. That is to say, hydrogen bonds are formed with water molecules to form what is called a “beta sheet”. This beta sheet can be thought of as ribbons of amino acids that stabilize themselves, via structure, in water, through hydrogen bonding.

The sequence of the 238 amino acids is GFP’s primary structure. The hydrogen bonding by these amino acids with water molecules is GFP’s secondary structure. Most every protein has a tertiary structure. The beta sheet of GFP folds back upon itself to form what is called a “Beta Barrel.” This formation is again driven by the interaction of the protein with itself and the water environment. Remember, all these amino acids have distinct side chains, and these side chains have distinct chemistries. So in the tertiary structure, the protein folds to form a structure to make sure all of the amino acid side chains that like water face outwardly and interact with water, and side chains that do not like water are shielded from water. So for GFP, looking at the beta barrel illustration above, the water hating part is shielded because it is located between “ribbons”and thus, shielded from water. The water loving part is hanging off the ribbons, interacting with water (this is an oversimplification, but gener-ally works). As a final note, the ribbons could have folded up into any structure, but the beta barrel is the most energy efficient structure given GFP’s primary structure (sequence of amino acids). Nature is elegant.

If you remember nothing else, just know that the GFP is a beta barrel with a chromophore protected in the center of the bar-rel. The chromophore in this case is formed via an interaction between amino acid #65 (Serine 65), Tyrosine #66, and Glycine #67 that absorbs blue light and emits green, giving GFP its green glow.

You can create different chromophores to GFP and have the protein emit different colors of light. Think red, blue, green, yel-low, etc., any wavelength that is visible. So the big picture goes like this:

1. You can create any visible fluorescence you want, given changing the chromophore by changing amino acids 65, 66, or 67, or some combination of the three.

2. You can link green fluorescent proteins to other molecules.

3. You can link green fluorescent protein variant that emits color x,y, z, etc. to any molecule.

4. Build a detector that looks for a molecule via colored light, and now you can do some targeted science!

A simple example is DNA sequencers. DNA is made up of A,C,T,G base pairs. Tag each molecule with green, blue, red, or yel-low, and then you can determine the sequence of any DNA mol-ecule based upon the color pattern. Link GFP to a drug that targets cancer cells, and then inject the drug into biopsied tissue. If the drug lights up in cancerous cells, but not normal cells, you have success. The drug might or might not work, but at least you know the drug is targeting the bad cells and not the good cells. The applications are limitless. Fluorescenceimageofgrayfoxlungfibroblast.a)Transfectionb)tran-

scriptionc)translationd)traffickingandlocalizatione)incorporationintoactinfilaments.Scalebaris10microns.

APPENDIX2

38 |

Appendix 3 - Fluorescence

When a substance emits light after absorbing light or other electromagnetic radiation of a different wavelength, the process is called fluorescence. In most cases, the emitted light has a lower energy (longer wavelength). When an atom absorbs radiation, an electron may be excited and move to a higher energy level. When that atom returns to its lower energy (or ground state), a photon (or packet) of light is emitted (see illustration above right). This illustration shows the more simplified Bohr model of electron orbitals, but helps demonstrate the general idea of fluorescence.

Fluorescence may also occur outside the visible spectrum, and has a wide variety of uses within many scientific fields. Many scientific uses take advantage of fluorescence within the visible spectrum, usually caused by absorption of ultraviolet (UV) light (see illustration above left). The growing field of fluorescence microscopy has led to many discoveries about processes which are otherwise very difficult to visualize. Fluorescent substances can be introduced into an organism and made to fluo-resce, allowing visualization of processes within cells while an organism is still alive.

lightlight

Sulfur

Endothelialcellstaggedwithavarietyoffluorescentmarkers,viewedwithafluorescentmicroscope.


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Appendix 4 - Chemistry of GFPOnce you understand the general structure of GFP (see Appendix 2), it is important to understand the chemical reactions in-volved in making GFP glow. How is a chromophore formed? Chemically, it involves a multi-step reaction. In the illustration below, the chromophore is shown in stick representation, with carbon in gray, oxygen in red, and nitrogen in blue. Heim et al. (1994) suggested this mechanism for chromophore formation: after translation of the protein takes place, a series of modifica-tions converts the serine 65, tyrosine 66, and glycine 67 tripeptide sequence into a fluorescent chromophore. The final appear-ance of the chromophore is show in part A. In part B, dashed lines represent connections to the polypeptide chain.

To achieve the variation in color between different chromophores, slightly different chemicals are needed. Each chromophore shown below represents a different source or a different color:

A. Aequorea victoria’s green fluorescent protein chromophore (Shimomura, 1979).

B. Discosoma (a coral) red fluorescent protein chromophore.

C. Zoanthus (a marine mat formed of individual polyps) yellow fluorescent protein.

D. Anemonia sulcata (an anemone) fluorescent protein chromophore

E. Trachyphyllia geoffroyi (open brain coral) red fluorescent protein chromophore.

F. Two stereoisomers of fluorescent protein chromophores.

APPENDIX4

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