Pglo Transformation Student Manual

Bacterial Transformation Efficiency. Difficulty. Time Required. Average (6- 1. 0 days)Prerequisites.

Some laboratory experience required: knowledge of sterile technique, working with bacterial cultures, and using automatic pipets all helpful. Material Availability. Specialty items. Cost. Average ($5. 0 - $1. Safety. Requires adult supervision in a laboratory facility.

For ISEF- affiliated fairs, this project will require SRC approval. Abstract. Is it possible to manipulate bacteria to become protein production factories? Can diabetics control blood glucose with insulin produced by bacteria? How cool would it be to take advantage of these microorganism's sophisticated makeup, short doubling times and cheap growth media to mass produce medically and commercially useful proteins? All of these are possible with a few simple genetic manipulations. By the end of this project you would know the basic foundation on which many biotechnology and pharmaceutical companies operate. Objective. The goal of this project is to measure bacterial transformation efficiency as a function of plasmid DNA concentration.

Credits. By Elizabeth Umelo- Njaka, Ph. D. Edited by Andrew Olson, Ph. D., Science Buddies. Cite This Page. MLA Style.

Science Buddies Staff. Bacterial Transformation Efficiency". Science Buddies. Science Buddies, 2. Oct. 2. 01. 4. Web.

Jan. 2. 01. 6. < http: //www. Bio. Chem_p. 01. 3.

Biotechnology Explorer™ pGLO™ Bacterial Transformation Kit Catalog #166-0003EDU explorer.bio-rad.com For technical support call your local Bio-Rad office, or in. The Student Manual is the official statement of University policies and regulations, and expected standards of student conduct that are applicable to all students. Seminary, seminary manuals, seminary videos, scripture mastery, scripture mastery games. AP Lab 4- Water Potential Student Answer Sheet : McGraw Hill Animations. AP Lab 7- Environmental Effects on Mitosis. Learn biotechnology techniques first hand while discovering the relationship between bacterial transformation efficiency and plasmid DNA concentration.

Demonstrate the power of genetic transformation! Students will glow with excitement when they transform bacteria with pGLO plasmid. Ideal for AP Biology Lab 6. LabBench Activity Molecular Biology. by Theresa Knapp Holtzclaw. Introduction. In this laboratory you will use some basic tools of molecular biology to gain an.

Science Buddies Staff. October 2. 2). Bacterial Transformation Efficiency. Retrieved January 2. Bio. Chem_p. 01. 3. Last edit date: 2.

Introduction. Bacteria are biochemical powerhouses, completely self- contained with all that is needed to produce complex proteins. Without microscopes, the human eye only begins to see bacteria when they have multiplied to literally millions of identical copies all in one spot, called a colony. The DNA molecule is the blueprint for every component of the bacteria. From information in the DNA, RNA molecules are transcribed and then translated into proteins. The proteins are moved to different parts of the bacteria—cytoplasm, periplasm, or cell wall—depending on function. Bacteria have transport systems that shuttle these proteins around, and sometimes there may be more than one type of transport system.

There are several ways in which bacteria acquire foreign DNA, including the processes of conjugation, transfection, and transformation. Conjugation involves mating between two different bacterial cells. In transfection, viruses called bacteriophages inject the foreign DNA into their host. In transformation, bacteria take up DNA from the environment through their cell wall.

Troubleshooting There are many reasons for variations from expected results. Some factors that can influence transformation efficiency include the type bacteria used.

Natural transformation was discovered in 1. Frederick Griffith while studying infectious bacteria that cause pneumonia in mice. Griffith was using two strains of pneumococcus bacteria: a virulent, smooth strain and a non- virulent, rough strain. On injecting mice with a mixture of killed smooth strains (which were incapable of causing infection) and the non- virulent rough strain, miraculously (the miracle being what is now known as transformation), the mice died and Frederick recovered live smooth pneumococcus bacteria! Inside the mice, the piece of DNA from the killed smooth strain containing the information required to cause infection had been taken up by the live, non- virulent, rough pneumococcus bacteria. In this way, the formerly non- virulent bacteria acquired the virulence traits and became deadly to mice.

In Frederick's control experiments, mice exposed to only rough cells stayed nice and healthy and those exposed to the dead smooth cells also stayed nice and healthy. Only when the live rough bacteria and killed smooth bacteria were administered together did the mice become sick. Why Is Bacterial Transformation So Important and Why Does It Hold Such Attraction to Science? Transformation in and of itself is a very important basic tool in molecular biology. Transformation is used for cloning or to move DNA molecules around between strains. Bacteria are transformed for numerous different reasons.

Some of these reasons may include expression of medically useful recombinant proteins such as insulin for treating a disease or vaccines for prevention of disease. Other reasons could be expression of proteins that confer on bacteria the ability to survive in particular environments such as to "clean up" contaminated environments in bioremediation. It can be very expensive to chemically synthesize very short peptides, never mind complex polypeptides and whole proteins which may have post- translational modifications. As the biology of bacteria becomes clearer, coupled with the abundance of bacterial species and strains available and the exciting advances made in molecular biology research and biotechnology, the possibilities and applicability of transformation becomes phenomenal.

How Does It Work? Not all bacteria undergo natural transformation. However, a large number of strains and species can be artificially transformed. In the laboratory when transformation occurs, the bacteria acquire new genetic traits (for example, resistance to a specific antibiotic) which are easily identifiable and allow for selection of transformed cells. Before bacteria can be artificially transformed, they have to be made competent—able to take up DNA. The DNA molecule is hydrophilic (water- soluble) but cell membranes are made of a very hydrophobic lipid bilayer, and therefore artificial transformation is not a process that occurs spontaneously.

There are two means of artificial transformation commonly used in labs: electroporation and chemical transformation. During electroporation, short bursts of current are passed through a solution containing bacteria at high voltage.

The current makes the cell membrane leaky (porous) for a short time, allowing the cells to take up DNA molecules from the solution. In chemical transformation, bacteria are exposed to solutions which alter their cell membranes enough to make the DNA molecules pass through and into the cell. Chemical transformation procedures sometimes also use a heat shock treatment. The actual mechanisms by which these two processes work are not fully understood. Transformation efficiency is a measure of the amount of cells within the bacterial culture that are able to take up DNA molecules. Transformation efficiency can be determined experimentally.

For some molecular biology projects, such as cloning and subcloning, high transformation efficiency is not critical. However applications such as construction of genomic libraries require that the bacteria have very high transformation efficiency. When bacteria are transformed in the laboratory, the bacteria acquire new traits from the transformation plasmid. These traits are easily identifiable and allow for selection of transformed cells. For example, the bacteria transformed in this project acquire resistance to the antibiotic ampicillin. You prove this by growing them up on LB: AMP plates. Untransformed bacteria will not grow on the LB: AMP plates.

Sometimes, scientists get really creative and add genes for other traits to these plasmids. Take the p. GLO plasmid from the Bio- Rad p. GLO transformation kit as an example. In addition to containing the ori gene which is essential for the plasmid to replicate inside the bacteria, and the Ampicillin resistance gene (bla gene) used for selection of transformed cells, it also contains the gene that encodes the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria. The GFP gene is placed under the regulation of the arabinose promoter. Because bacteria are highly economical and tend not to waste energy, they have different types of promoters which regulate gene expression. For those proteins which are required only under specific conditions, the genes that encode them are turned off when not needed to conserve energy and turned on only when the bacteria encounters the right conditions.

For example, the genes which encode the proteins required to break down and use the sugar arabinose are turned on only in the presence of arabinose. Therefore by placing the GFP protein gene under the control of the arabinose promoter, scientists can selectively express the GFP protein by including or excluding arabinose from the growth media. This allows for increased utility of the p. GLO plasmid not just simply for cloning, but also for in situ visualization of protein expression because those bacteria that have taken up the p. GLO plasmid will turn a brilliant green color (as shown in Figure 1, below) when they are grown in a media containing arabinose.

Figure 1. These bacteria are expressing GFP. Photo courtesy of Bio- Rad Laboratories, Inc.). Terms and Concepts For full appreciation of this experiment, you should understand the following terms and concepts: Bacteria Transformation Transformation efficiency Cloning and cloning vectors Antibiotic resistance DNAses, restriction endonucleases and ligation Heat shock and cold shock Promoter Regulation of gene expression Aseptic or sterile techniques Biotechnology. Questions What is meant by the term competent when applied to bacteria being prepared for transformation? What is the relationship between genes and DNA? What is the relationship between DNA and RNA? What is the relationship between RNA and proteins?

Bibliography This website provides access to publications on cloning and bacterial transformation: NCBI, 2. Education," National Center for Biotechnology Information [accessed October 2.

Education/. This is a simplified version of the classical method used in this transformation exercise (see the Variations section): Chung, C. T., S. L. Niemala, and R. H. Miller, 1. 98. One- step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution," PNAS 8.

October 2. 0, 2. 00. Adobe Acrobat). This site provides access to many molecular biology protocols: Open. Wet. Ware contributors, 2. Main Page," Open. Wet. Ware [accessed October 2. At the Bio- Rad website, you can download the complete instruction manual for the p. GLO transformation Kit.

The manual contains lots of useful information including diagrams and protocols (requires Adobe Acrobat). In order to download literature, you must register at the Bio- Rad site. GLO" in the "Literature" section of the search box.: Bio- Rad, 2. GLO Bacterial Transformation Kit Instruction Manual," Bio- Rad, Inc. October 2. 5, 2. 00. Materials and Equipment Note: If you are carrying out this experiment in a school laboratory, which is recommended, some of the materials and equipment listed may be more readily accessible.

The p. GLO Bacterial Transformation Kit from Bio- Rad is recommended however, any. The p. GLO transformation kit contains. DNA, reagents, etc.

A teacher's help will be needed when. GLO kit as Bio- Rad only sells directly to schools. Permanent marker Ice bucket with ice. You can substitute styrofoam cups with ice. C incubator. Alternatively, bacteria will grow at room temperature but it takes days longer. C water bath. Alternatively, hot water from the tap is sufficient; use a thermometer to ensure the temperature is exactly 4. C for the 5. 0 second duration of the heat shock.

Automatic pipettors in 1–1. L and 1. 0–2. 00 μL ranges. Graduated pipets supplied with the Bio- Rad kit can be used instead if unavailable. Thermometer (0–1. C), available from online suppliers such as Carolina Biological Supply Company. Clock or watch to time 5. L distilled water; usually available from a supermarket Microwave oven.

This is needed to prepare the agar plates. Refrigerator. This is for storing plates before use. UV- protective face shield or goggles for visualizing p. GLO. UV- protective goggles are available through Carolina Biological. Note: Bio- Rad Kits are sold directly to schools. Please visit this page to learn how to purchase one. Science Buddies occasionally provides information (such as part numbers, supplier.

The. information is provided solely as a convenience to our users. We do our best to make sure that part numbers.

However, since part numbers do change as items are obsoleted. We also do our best to make sure that any listed supplier provides prompt, courteous service. Science Buddies does participate in affiliate programs with. Carolina Biological, Jameco Electronics.

Aqua. Phoenix Education. Proceeds from the affiliate programs help support Science Buddies, a 5. If you have any comments (positive or negative) related to. Write. to us at scibuddy@sciencebuddies.

Experimental Procedure. Working with Biological Agents. For health and safety reasons, science fairs regulate what kinds of biological materials.

You should check with your science fair's. Scientific Review Committee before starting this experiment to make sure your science. Many science fairs follow Intel®.

International Science and Engineering Fair (ISEF) regulations. For more information. Science Buddies pages.

Projects Involving Potentially Hazardous Biological Agents and. Scientific Review Committee.

You can also visit the webpage. ISEF Rules & Guidelines directly. Do your background research so that you are knowledgeable about the terms, concepts, and questions above. Read the product insert from the transformation kit prior to starting your experiment. You need to understand the sequence of the experimental protocol and prepare materials accordingly.

Follow the directions provided in the kit. Note that you will include an additional step to determine how varying the concentration of DNA affects transformation efficiency.

The following procedure is based on the assumption that the Bio- Rad p. GLO transformation kit is used. Day 1: Preparing Plates, Solutions, and Bacterial Starter Plate. Follow the kit directions to prepare and pour the agar plates, and to rehydrate the provided lyophilized materials such as E. DNA, etc. Prepare and pour the agar plates—LB only (LB) and LB plus ampicillin (LB: AMP).

Label the plates with permanent marker: LB and LB: AMP. After the agar solidifies, cover the plates, put them in their original plastic bags, and store them in a lab refrigerator stacked upside down. Store plates wrapped up in their original plastic wrappings. Storing upside down will ensure condensation does not wet the surface of the agar. Note that the p. Glo Transformation kit also allows for visualization of the transformation. In addition to the acquisition of Ampicillin resistance, the transformed bacteria can also express another gene on the p. GLO plasmid which causes the bacteria to glow a brilliant green color.

In order to see this, prepare the LB: AMP: ARA agar plates as specified in the p. GLO transformation kit product insert. After transformation on day 2, plate the transformed cells on the LB: AMP: ARA plates as well.

The arabinose in the agar will induce expression of the green fluorescent protein and the bacteria will glow green. While this step is cool to see it is not required for you to determine transformation efficiency. The Bio- Rad p. GLO transformation kit comes with one UV penlight. This should be sufficient to visualize the glowing bacteria.

However if you have access to a laboratory with a long wave UV lamp, that will be great. Caution - Do not shine UV light directly into the eyes, use a UV- protective face shield or goggles, and limit exposure to UV light.

Rehydrate bacteria and streak LB starter plates. Incubate starter plates overnight at 3. C (or 2 to 3 days at room temperature until colonies are clearly visible).

Day 2: Transforming the Bacteria. Important: do not refrigerate the starter plate prior to transformation. Label 3 tubes with the following. GLO plasmid (negative control), +1× p. GLO plasmid, +1. 0× p. GLO plasmid. Using a graduated pipette add 1 m.

L of the transformation solution to a clean tube. With a sterile loop, choose 4 well- separated colonies from the starter plate and resuspend in the tube containing 1 m. L of transformation solution by flicking the tube or by twirling the loop around in the solution. Note that each colony contains millions of bacterial cells—do not use too many colonies). When the colonies are completely resuspended, use a clean graduated pipette to transfer 2. L of the bacterial solution to each of the 3 tubes labeled −p. GLO plasmid, +1× p.

GLO plasmid, and +1. GLO plasmid. Add 1.

L of the p. GLO plasmid solution to the tube labeled +1× p. GLO plasmid. (If you don't have access to automatic pipettors, 1. L is one sterile loop full.) Add 1. L of the p. GLO plasmid solution to the tube labeled +1. GLO plasmid. Mix by covering and flicking the tubes.

Do not add p. GLO plasmid DNA to the −p. GLO plasmid (negative control) tube. Incubate the 3 tubes on ice for 1. Heat shock the 3 tubes at 4.

C for 5. 0 seconds exactly. Immediately place tubes on ice for 2 minutes. Add 2. 50 μL of LB broth to each of the three transformation tubes and incubate at room temperature for 1. Use a clean pipette for each addition. Spread 1. 00 μL of bacterial suspension on to the appropriate LB: AMP plates, use 2 plates per transformation. Use the average number of colonies from the two plates in subsequent calculation.

Incubate the plates overnight at 3. C (or at room temperature for 2 to 3 days). Day 3: Analyzing and Interpreting Results Count the average number of colonies growing on the 2 LB: AMP plates.

Determine the average amount of DNA that was spread on the plates. Use the examples to determine the average amount of DNA spread on the plates. Total DNA supplied in tube = 2. Volume of transformation solution added to reconstitute = 2. L. Concentration of DNA solution = 2.

L (or 0. 0. 8 μg/μL). For the 1. X transformation, 1. L of the DNA solution is added into 2. L of bacteria resuspended in transformation solution, so the total volume in the tube = 2. L, and the total DNA concentration in the tube = 1. L × 0. 0. 8 μg/μL = 0. DNA/2. 60 μL. After transformation, 1.

L is added to each plate, so the amount of DNA plated = (0. L/2. 60 μL) × 1. 00 μL = 0.

For the 1. 0x transformation, 1. L of DNA solution is added into 2. L of bacteria solution, so the total volume in the tube = 3. L, and the total DNA concentration = 1. L × 0. 0. 8 μg/μL = 8.

L. After transformation, 1. L is added to each plate, so the amount of DNA plated = (8.

L) × 1. 00 μL = 2. Calculate transformation efficiency for the 1. X and 1. 0X DNA concentrations using the formula.

Which experiment had greater efficiency? Bacterial Safety. Bacteria are all around us in our daily lives and the vast majority of them are. However, for maximum safety, all bacterial cultures should always be.

This means that proper handling, cleanup, and disposal. Below are a few important safety reminders. You can also see the. Microorganisms Safety.

Guide for more details. Additionally, many science fairs follow. ISEF Rules & Guidelines, which have specific guidelines on how bacteria. Keep your nose and mouth away from tubes, pipettes, or other tools that come in. Make sure to wash your hands thoroughly after handling bacteria. Proper Disposal of Bacterial Cultures. Bacterial cultures, plates, and disposables that are used to manipulate the bacteria.

Use caution when handling the bleach, as it can ruin your clothes if spilled, and. After bleach treatment is completed, these items can be placed in your normal household. Cleaning Your Work Area. At the end of your experiment, use a disinfectant, such as 7. Be aware of the possible hazards of disinfectants and use them carefully.

Variations. What happens to transformation efficiency when you try the following: Transform E. Try the one- step transformation protocol (Chung, Niemala, and Miller, 1. Experimental Procedure section? Use stationary phase culture compared to log phase? Compare chemical transformation to electroporation using identical concentrations of DNA and bacterial cells?

Heat shock for various lengths of time, e. How does efficiency change with the length of the heat shock? Why? Hide feedback on this project from other users. Recent Feedback Submissions. Carleigh. Gillaspie. What was the most important thing you learned? You learn so much!

What problems did you encounter? Price, but it was not to bad. Can you suggest any improvements or ideas?

Not at all. loved it. Overall, how would you rate the quality of this project? Excellent. What is your enthusiasm for science after doing your project?

Very high. Compared to a typical science class, please tell us how much you learned doing this project. Much more. 2. 01. What was the most important thing you learned?

I learned that the more bacteria you insert into a cell, it could affect the growth of the bacteria or even overwhelm the cell. What problems did you encounter? The biggest problem in this experiment was finding a lab and a mentor. After over 5. 0 emails, I got acceptance from a microbiologist. Can you suggest any improvements or ideas?

If you do this experiment, make sure to do more than 1 trial. Also test if heat shock effects the experiment also. I only used 1 trial, so I don't know if my results were accurate or not. So in the future that's what I could look into.

Also if you would like to do this experiment, use all the concentrations ranging from 1 to 1. In my experiment, I only used 1 and 1. So In the future, I can test the optimal range of concentration. Overall, how would you rate the quality of this project?

Excellent. What is your enthusiasm for science after doing your project? Very high. Compared to a typical science class, please tell us how much you learned doing this project.

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How do plants respond to ultraviolet light? Beyond basic research, biologists might also apply their research and create new biotechnology. There are endless discoveries waiting to be found in the field of biology! Read more 0">. News Feed on This Topic. Note: A computerized matching algorithm suggests the above articles. It's not as smart as you are, and it may occasionally give humorous, ridiculous, or even annoying results! Learn more about the News Feed.

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