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IT Report # 1 Due Dec 21 / 98 AN EXPERIMENT IN PROPHAGE MANIPULATION: Submitted By: Jeff Scott # 968468 IT Report # 1 AN EXPERIMENT IN PROPHAGE MANIPULATION: An Experiment in Prophage manipulation, INTRODUCTION: Numerous bacteriophages have been identified, which integrate their DNA sequences into the host cells chromosome (the result is called a propane). If this occurs, the host along with its new chromosome structure is defined as a lysogen. This is so, because the act of a bacteriophage which successfully integrates its viral genes into the hosts chromosome without killing the host si defined as lysogeny. Lysogenic bacterial cells are very hard to detect because of the fact that the viral genes will function quiescently.
Furthermore, lysogenic cells do not produce some essential proteins, especially if the propane covers essential genes of the host. In this profile I will attempt to show certain methods that can be used to map prophage's, as well as show some possible techniques one might use to eliminate the prophage's influence on the host cells functions. PROBLEM: A very unique strain of E. coli bacteria, 186 sen, has been isolated from pond samples taken in the low latitude tropical rain forests of the Amazon drainage basin in South America. 186 sen was named according to preliminary experiments with page sensitivity.
This bacterial strain is unique in that it synthesizes a very effective gram positive antibiotic, which if isolated could be the answer to eliminating many bacterial decease (like Strep throat or food poisoning via Staphylococcus aureus etc) which have been gradually building remittances to the current antibiotics being used. As the name implies, this bacteria is very sensitive to lysogenic phage induction via the 186 phage (believe it or not, this is an actual type of phage). The task before us is to An Experiment in Prophage manipulation, devise experiments we can use to possibly overcome the lysogenic effects this particular virus has on this particular strain of E. coli.
HYPOTHESIS: One possibility of deceasing the lysogenic effect of this phage would be to map the inserted viral genes. After which we could use specialized restriction endonuclease's to cut the genes on either side. Then test the bacteria to see if any of the essential genes, for the production of antibiotic, lie within the propane region. EXPERIMENT # 1: Step 1: Before we attempt any lengthily experiments, we must first determine weather or not the bacteria is even infected.
To do this we super infect the with the 186 phage. If the phage is immune to the super infection we know it is already a lysogen, and can therefore proceed (refer to figure 1). An Experiment in Prophage Manipulation, Step 2: Using an Her contra selected marker tet 2 which maps at a known position of 84. 5 min on the E. coli linkage map, we can now transfer the infected bacterial genes into an uninfected 186 sen cell, using Her conjugation techniques (refer to figure 2). To see which side of the tet 2 marker the propane lies, we can use marker rescue (refer to figure 3).
An Experiment in Prophage Manipulation, After crossing over all the genes from 0 min to 85 min (just ahead of the tet 2 marker) we can now super infect the recombinant cell to see if the propane genes lie within this area on the host gene (refer to figure 4). The super infected recombinant cell shows the integration of the viral genes from the 186 phage. Therefore the cell is not immune to super infection, which tells us that the viral genes are inserted after the 84. 5 min mark on the E. coli linkage map.
In order to be sure, we will do the same Her experiment, only this time the marker rescue must occur between the 84. 5 min mark and the 100 min mark. By super infecting this exconjugant we see it is immune to the phage induction. This assures us that the propane indeed lies between the 84. 5 min and the 100 min marks on the E. coli linkage map. NOTE: Similar experiments can be done to see if 186 phage is in the bacterial cells chromosome using the method of Zygotic Induction. But I find using super infection much easier.
Also, there was no sign that the 186 phage immunity was located in the cytoplasm. EXPERIMENT 2: We can further map the 186 prophage's location using P 1 transduction. We know that the tet 2 marker lies at 84. 5 min on the E. coli linkage map.
And we also know that the 186 propane lies between 84. 5 and 100 min. [use your imagination, because Im not going to bother wasting yours or my time writing out a pretend P 1 transduction experiment] The results from the P 1 transduction show that there is a 67 % co transduction frequency between the marker tet 2 and the 186 propane (how convenient). This data tells us that these two genes are quite close together (refer to figure 5). Given the previous data, and knowing that the tet 2 marker can be cotransduced with the 186 propane (which infers that they are within 2 min of each other on the E. coli linkage map).
And also knowing that the 186 propane is on the right side (ie between 84. 5 and 100 min) we can now with a good degree of accuracy tell where the 186 propane is located on the chromosome. EXPERIMENT 3: Using specifically engineered endonuclease's which cut at precise origins on the E. coli chromosome, we can cut the 186 propane out (refer to figure 6). After the suspected propane region is cut out and the subsequent ends are joined by our own synthetic DNA ligase molecule, we can now test the remaining gene to see if any of its essential genes were covered by the 186 propane.
To do this we introduce the new gene product into an environment (probably in vitro) containing active gram positive bacteria and observe the results. As we had hoped, the results after a 48 hour incubation showed no sign of the gram positive bacteria. Which tells us that the 186 propane, when mapped properly, can be cut out of the E. coli gene without taking out any essential genes needed to produce the effective gram positive antibiotic. CONCLUSION: The relevance of this experiment is due to the growing problem of antibiotic resistance that many previously treatable pathogenic bacteria are developing. Another factor is the over use of the present antibiotic arsenal, as well as the fact that no new antibiotics have been discovered for many years.
Given these facts along with the remarkable genetic diversity and adaptability which bacteria show, its only a matter of time until we will have no defences. Thats why research in this field is so important to mankind.
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