GLOW-IN-THE-DARK BACTERIA (Transformation Lab) A. BACKGROUND INFORMATION The Problem of Infectious Disease: At the turn of this century, infectious diseases like pneumonia and influenza caused high numbers of deaths, particularly among children. Accidents and war also caused body tissue infections that resulted in deaths among soldiers and other adults. By the late 1800's, scientists had shown that vaccinations could prevent some illnesses like smallpox, and that you could cure some surface infections with disinfectants. As a result, many governments and private companies in Europe and the United States funded an all-out effort to find way to prevent or cure infections. This is the context in which scientific discoveries regarding bacteria were viewed. The Quest for Disease Prevention: Throughout the 20th century, many scientists worked on developing vaccines against all types of human infections. As all of know from the number of doctor visits and shots we have had, this effort was very successful - we now have preventative vaccines against most bacterial and viral diseases. The quest for vaccines also led to other scientific discoveries. In 1928, one such scientist named Frederick Griffith was working on this problem of finding a vaccine against pneumonia caused by the bacteria Streptococcus pneumoniae. Here s what he found: In Experiment A, Griffith injected mice with a deadly strain of pneumococcus that had a capsule. When Griffith removed the capsule in Experiment B, all the injected mice lived. Griffith then heat-inactivated the deadly capsulated pneumococcus for Experiment C, and all the injected mice survived. Finally, in Experiment D, Griffith mixed live harmless pneumococcus with heat-killed capsulated pneumococcus. While neither of these strains killed mice alone, they did in combination!
While Griffith didn t directly succeed in making a vaccine, he did discover that bacteria can transform themselves into new species by absorbing DNA from dead bacteria. He also discovered, much to his dismay, that the S. pneumoniae bacteria transformed in a way that made them more infectious to humans Searching for a Cure to Infection: About this same time, in England, Alexander Fleming was trying to solve the problem of curing infectious illnesses another way - by searching for natural compounds (antibiotics) that could kill bacteria without harming people. He succeeded - he found a mold called penicillium produced a natural antibiotic (penicillin) that would kill many bacteria. These two scientific discoveries - antibiotics and bacterial transformation - both began as part of man s quest to prevent and treat infections. We now find that antibiotics and transformation are once again linked together. And this inter-relationship has posed a new health hazard for humans. Here s how: Alarming Evolutionary Relationship Between Transformation and Antibiotics: With the increased availability of antibiotics, doctors can now treat almost every infectious illness. However, as the use of antibiotics became common, bacteria adapted to the presence of antibiotics in humans, and bacteria have undergone DNA mutations that allowed them to resist the effect of antibiotics. That s where transformation comes back into the picture - when a bacteria developed mutations to resist antibiotics it could pass on this characteristic to other bacteria through the natural process of transformation. As a result, research scientists fear that bacteria are now transforming into antibiotic-resistant strains almost as fast as we can develop new antibiotics. Transformation - Nature s Own Genetic Engineering: In nature, bacteria can transfer genes (via plasmid DNA) between two different bacteria strains or species. When a bacteria dies, its plasmid DNA can be taken in by other bacteria living in the area. The live bacteria thereby acquires new genes that it can use. In nature, only a few species of bacteria are competent to take in stray plasmids, due to chemicals they make called competence factors. As a result, the incidence of transformation in a natural setting is relatively random and infrequent. However, scientists have developed controlled chemical and heat/cold conditions that increase the rate of transformation in a lab setting. Here is how the laboratory transformation process works to produce a bacteria that is transformed to be resistant to a certain antibiotic:
Tranformation: The Foundation of Genetic Engineering: By the 1970's, scientists knew that DNA structure and codon signals were similar for all organisms. They also had developed reliable procedures for transformation. Berg and Cohen then realized that the natural process of transformation could be used to create new, artificial organisms that combined genes from different species. The rest is history - and the biotechnology industry was born. Today, we make hundreds of medications and genetically modified plants and animals using the basic process of transformation. In this lab, we will see firsthand how bacteria can absorb DNA from an outside source, and how that foreign DNA can allow the bacteria to become resistant to antibiotics and acquire other traits. In the course of our study, we will: 1. Learn sterile technique for handling bacteria 2. Learn to use micropipets. 3. Perform a transformation experiment. Our E. coli bacteria will start out sensitive to antibiotics and have normal pale yellow coloration. We will then add a P-GLO plasmid containing bacterial genes that code for resistance to the antibiotic ampicillin and a jellyfish gene that codes for green fluorescent protein. Our original plain E. coli bacteria will then transform to become antibiotic resistant and produce glowing colonies. 4. Calculate the transformation efficiency with P-GLO and compare that efficiency rate to other plasmids and compare efficiencies for different strains of bacteria. B: DETAILS ABOUT OUR TRANSFORMATION PROCEDURE The plasmid: We will purchase genetically-engineered P-GLO plasmid and others, and will use them to transform our plain E. coli bacteria. Making our E. coli competent : We use series of heat and cold shocks, combined with a certain concentration of salt (calcium chloride) to disrupt the bacterial cell membrane and make it competent. Without these procedures, the bacteria will not take in the plasmid. Selecting transformed bacteria: Transformation, even in a lab setting, is not perfect. Most of the bacteria will not take in the plasmid. Therefore, we must have a way to separate the cooperative transformed bacteria from the non-transformed. We use the antibiotic ampicillin for this purpose. Transformed bacteria will survive ampicillin, but the non-transformed bacteria will not. So, after the experimental procedure, we feed ampicillin to the bacteria to kill any non-transformed cells. Jellyfish protein and arabinose genes: Remember that most genes are turned off, and must be switched on to be transcribed. As a result, when scientists created the P-GLO plasmid, they had to put the jellyfish gene inside an inducible operon. The arabinose operon serves that purpose. In order to make the bacteria glow green, we must feed them arabinose - it induces the genes in the operon to turn on. A diagram of the arabinose operon and GFP gene is shown below.
Original Arabinose Operon Contains control gene (AraC) and three digestive enzyme genes (Ara A, Ara B, Ara D). If arabinose sugar is present, it will act as an inducer to the operon: it will bind to AraC and cause the three enzyme genes to be transcribed. Genetically Engineered Operon : GFP gene inserted and arabinose digestive enzyme genes are deleted. Arabinose acts as an inducer for the operon. When fed arabinose, the bacteria will transcribe genes in the operon and make GFP. C. OBSERVING, GROWING AND HANDLING BACTERIA Safe Handling of Bacteria: Bacteria are present in almost every type of environment. They serve a number of useful purposes, such as nitrogen fixation in plants, recycling of oxygen, recycling of CO 2 during decomposition, sewage treatment and production of food items such as yogurt and cheese. Bacteria also, unfortunately, can cause diseases in other living organisms. Principles of Sterile (Aseptic) Technique: Because some bacteria can cause illness, and because stray bacteria can easily get into our experiment, we must be careful in how we handle our bacteria specimens and our experimental equipment and supplies. Microbiologists have developed safe handling techniques, called sterile technique or aseptic technique, to minimize the risk of becoming infected or contaminating our lab. The three basic goals of using sterile technique in our experiments are: 1) Avoid contaminating the work place, 2) Do not contaminate yourself while handling bacteria and 3) Do not contaminate the experiment. To accomplish sterile technique, we follow these rules: Sterilize instruments with heat.. Clean work surfaces with 10% bleach (a strong disinfectant). Keep bacteria cultures covered to avoid contamination. Dispose of bacteria-contaminated equipment in special bio-hazard garbage bags. Clean up bacteria cultures with 10% bleach. Wash hands frequently, both before and after experiment. In the event of a spill of a live bacterial culture, remain calm and do the following: a. Report the accident immediately to the teacher. b. Cordon off the area and cover the spill with paper towels. c. Pour full-strength bleach liberally over the towels. d. After 15 minutes, dispose of the towels in the biohazard bag. Wear protective eye goggles and/or gloves as required.
2. TRANSFORMATION PROCEDURE FOR P-GLO The basic procedure is shown in the pictogram below. Once you have counted your colonies on the plate, you will calculate the transformation rate for each bacterial species and plasmid used. The details of the calculations are found in the lab worksheet. Transformation Lab Procedure Summary