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Project Summary

Difficulty	8  –  10
Time required	Average (about one week)
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 ($50 - $100)
Safety	Requires adult supervision in a laboratory facility. For ISEF-affiliated fairs, this project will require SRC approval.

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Sponsored by generous grants from Bio-Rad and its Biotechnology Explorer program

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Abstract

Objective

The goal of this project is to measure bacterial transformation efficiency as a function of plasmid DNA concentration.

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.

Natural transformation was discovered in 1928 by 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 pGLO plasmid from the Bio-Rad pGLO 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 pGLO plasmid not just simply for cloning, but also for in situ visualization of protein expression because those bacteria that have taken up the pGLO plasmid will turn a brilliant green color when they are grown in a media containing arabinose.

Terms, Concepts and Questions to Start Background Research

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, 2006. "Education," National Center for Biotechnology Information [accessed October 20, 2006] http://www.ncbi.nlm.nih.gov/Education/.
• This is a simplified version of the classical method used in this transformation exercise (see the Variations section, below):
  Chung, C.T., S.L. Niemala, and R.H. Miller, 1989. "One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution," PNAS 86 (7): 2172–2175, available online [accessed October 20, 2006]: http://www.pnas.org/cgi/reprint/86/7/2172 (requires Adobe Acrobat).
• This site provides access to many molecular biology protocols:
  OpenWetWare contributors, 2006. "Main Page," OpenWetWare [accessed October 20, 2006] http://openwetware.org.
• At the Bio-Rad website, you can download the complete instruction manual for the pGLO 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, and then search for "pGLO" in the "Literature" section of the search box.:
  Bio-Rad, 2006. "pGLO Bacterial Transformation Kit Instruction Manual," Bio-Rad, Inc. [accessed October 25, 2006] http://explorer.bio-rad.com.

Materials and Equipment

To do this experiment you will need the following materials and equipment:

• The pGLO Bacterial Transformation Kit is recommended (catalog # 166-0003EDU, Bio-Rad Laboratories, http://www.bio-rad.com, list price $89.00, as of April 2009) however, any equivalent transformation kit can be used. The pGLO transformation kit contains the bacteria, plasmid DNA, reagents, etc.
  Note: Bio-Rad Kits are sold directly to schools. To purchase, please have your school contact Bio-Rad at 800-424-6723 to verify account information and to place the order for you. Existing accounts will have orders processed within a day, and establishing an account will take approximately 48 hours.
• In addition, you will need to have access to a laboratory with the following:
  • permanent marker,
  • ice bucket with ice (substitute styrofoam cups with ice),
  • 37°C incubator (bacteria will grow at room temperature but it takes days longer),
  • 42°C water bath (hot water from the tap is sufficient; using a thermometer ensure the temperature is exactly 42°C for the 50 second duration of the heat shock),
  • automatic pipettors in 1–10 μL and 10–200 μL ranges (graduated pipets supplied with the kit can be used instead if unavailable),
  • thermometer (0–100°C),
  • clock or watch to time 50 seconds,
  • 1 liter flask,
  • 500 mL distilled water (bottled distilled water from supermarket is fine),
  • household bleach for clean up,
  • microwave oven (to prepare the agar plates),
  • laboratory refrigerator (for storing plates before use),
  • UV-protective face shield or goggles for visualizing pGLO.

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Experimental Procedure

1. Do your background research so that you are knowledgeable about the terms, concepts, and questions above.
2. Read the product insert from the transformation kit prior to starting your experiment.
   1. You need to understand the sequence of the experimental protocol and prepare materials accordingly.
   2. 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.
   3. The following procedure is based on the assumption that the Bio-Rad pGLO 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. coli bacteria, antibiotics, DNA, etc.

1. Prepare and pour the agar plates—LB only (LB) and LB plus ampicillin (LB:AMP).
   1. Label the plates with permanent marker: LB and LB:AMP.
   2. 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.
   3. Note that the pGlo 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 pGLO 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 pGLO 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 pGLO 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.
2. Rehydrate bacteria and streak LB starter plates.
3. Incubate starter plates overnight at 37°C (or 2 to 3 days at room temperature until colonies are clearly visible).

Day 2: Transforming the Bacteria

1. Important: do not refrigerate the starter plate prior to transformation.
2. Label 3 tubes with the following:
   1. −pGLO plasmid (negative control),
   2. +1× pGLO plasmid,
   3. +10× pGLO plasmid.
3. Using a graduated pipette add 1 mL of the transformation solution to a clean tube.
4. With a sterile loop, choose 4 well-separated colonies from the starter plate and resuspend in the tube containing 1 mL 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).
5. When the colonies are completely resuspended, use a clean graduated pipette to transfer 250 μL of the bacterial solution to each of the 3 tubes labeled −pGLO plasmid, +1× pGLO plasmid, and +10× pGLO plasmid.
6. Add 10 μL of the pGLO plasmid solution to the tube labeled +1× pGLO plasmid. (If you don't have access to automatic pipettors, 10 μL is one sterile loop full.)
7. Add 100 μL of the pGLO plasmid solution to the tube labeled +10× pGLO plasmid.
8. Mix by covering and flicking the tubes.
9. Do not add pGLO plasmid DNA to the −pGLO plasmid (negative control) tube.
10. Incubate the 3 tubes on ice for 15 minutes.
11. Heat shock the 3 tubes at 42°C for 50 seconds exactly.
12. Immediately place tubes on ice for 2 minutes.
13. Add 250 μL of LB broth to each of the three transformation tubes and incubate at room temperature for 10 minutes. Use a clean pipette for each addition.
14. Spread 100 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.
15. Incubate the plates overnight at 37°C (or at room temperature for 2 to 3 days).

Day 3: Analyzing and Interpreting Results

1. Count the average number of colonies growing on the 2 LB:AMP plates.
2. Determine the average amount of DNA that was spread on the plates. Use the examples below to determine the average amount of DNA spread on the plates:
   1. Total DNA supplied in tube = 20 μg.
   2. Volume of transformation solution added to reconstitute = 250 μL.
   3. Concentration of DNA solution = 20 μg / 250 μL (or 0.08 μg/μL).
      
      
   4. For the 1X transformation, 10 μL of the DNA solution is added into 250–μL of bacteria resuspended in transformation solution, so the total volume in the tube = 260 μL, and
   5. the total DNA concentration in the tube = 10 μL × 0.08 μg/μL = 0.8 μg DNA/260 μL.
   6. After transformation, 100 μL is added to each plate, so the amount of DNA plated = (0.8 μg/μL/260 μL) × 100 μL = 0.3 μg per plate.
      
      
   7. For the 10x transformation, 100 μL of DNA solution is added into 250 μL of bacteria solution, so the total volume in the tube = 350 μL, and
   8. the total DNA concentration = 100 μL × 0.08 μg/μL = 8.0μg/350 μL.
   9. After transformation, 100 μL is added to each plate, so the amount of DNA plated = (8.0 μg/350 μL) × 100 μL = 2.3 μg per plate.
3. Calculate transformation efficiency for the 1X and 10X DNA concentrations using the formula below.

4. Which experiment had greater efficiency?

Variations

What happens to transformation efficiency when you try the following:

• Transform E. coli with a small plasmid compared to large plasmid?
• Try the one-step transformation protocol (Chung, Niemala, and Miller, 1989) compared to the method described in the 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.g., from 25 s all the way up to 150 s. How does efficiency change with the length of the heat shock? Why?

• For more science project ideas in this area of science, see Biotechnology Project Ideas.

Credits

By Elizabeth Umelo-Njaka, Ph.D.

Edited by Andrew Olson, Ph.D., Science Buddies

Last edit date: 2009-01-05 12:00:00

Career Focus

If you like this project, you might enjoy exploring careers in Biotechnology.

	Biochemist Growing, aging, digesting—all of these are examples of chemical processes performed by living organisms. Biochemists study how these types of chemical actions happen in cells and tissues, and monitor what effects new substances, like food additives and medicines, have on living organisms.			Agricultural Technician As the world's population grows larger, it is important to improve the quality and yield of food crops and animal food sources. Agricultural technicians work in the forefront of this very important research area by helping scientists conduct novel experiments. If you would like to combine technology with the desire to see things grow, then read further to learn more about this exciting career.

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