Unadsorbed Phage Lab Report

The overall goal is to either discover a new novel phage that has not yet been discovered, or provide new research on an already discovered phage. The isolation of the phage takes several steps under the streak protocol that is present in materials and methods section of this report.

The main objectives of this experiment included making dilutions of solutions, plating phage or bacteria, and determining the number of bacterial viruses or phage in a suspension. It was also conducted to demonstrate that two different mutants of phage T4 can exchange genetic material to give rise to wild-type phage. The experiment was used to distinguish mutants from wild-type by their host specificity. The recombination in bacteriophage was performed to determine the concentration of unadsorbed phage from the U series plates, total concentration from B series, and concentration of

Second, the hoc protein found in the capsid of the T4 phage is only tested on agar plates. Although researchers are able to specify exactly what goes into the agar plate and the environment was replicated, the phage may react differently in the body due to other factors such as food consumption or medication usage. If this is true, the symbiotic relationship may still hold true, but there may be differences found on the mucus layers. Third, there may be other proteins that are involved in the phage that contribute to adherence to the mucus layer that were not uncovered in the study. Although the data seen from the hoc protein demonstrates that the hoc protein is involved based on the interaction between the mucin glycoproteins, the research is very specific to the hoc protein and there may be other proteins that are not found in the capsid that are involved in the defense mechanism.

Before plating the strains on agar plates, dilutions of the three strains of cells were prepared with LB broth.

typhimurium was ran on the first day to determine the CFU/mL of the overnight culture in order to calculate the volume of phage needed for transduction, the dilution of 10-8 was used since 93 colonies (9.3 x 109 CFU/mL) grew on the LB plate (See table 1). A titration of P22 phage and S. typhimurium were also performed to determine the PFU/mL of the P22 phage, a control plate with phage alone showed now growth, and the dilution 10-7 was found to have 132 plaques (1.32 x 109 PFU/mL). This value was used together with the dilution results of S. typhimurium on day 1 for calculating the volume of phage (see sample calculation). An MOI of 0.01 was used with these two values (volume of bacteria was .1mL) in order to determine the volume of phage needed for transduction, which ended up being 7µl. Using this information, three plates were run for the transduction, one plate with cells only on LB-CM, a plate with the cells and the phage on LB-CM, and a plate with phage only on LB-CM. There was only growth on the cells and phage plate, giving 158 colonies (1.58x103/mL) (see figure 1. And table 1. for results). This indicated successful transduction of the chloramphenicol gene since only colonies with the phage were able to grow on the antibiotic plate. A “final” titration was simultaneously run for the specific sample of recipient S. typhimurium actually used in the transduction, with the dilution of 10-8 resulting in 262 colonies (2.62 x 1010

The infection process of the T7 phage is generally the same as the two kinds of phages mentioned before. T7 phage also attaches the tail to the bacterium’s receptors and use lysozyme to degrade O or K antigens. This action creates an opening in the peptidoglycan cell wall and the genome is injected into the cell. A unique thing about the T7 phage is that it injects proteins that it injects proteins that help the replication of the viral genome and stops the duplication of the

Phage therapy involves the use of bacteriophages to treat specific pathogenic bacteria. The bacteriophages infect or kill pathogenic bacteria upon encounter with them. In 1915, Frederick Twort discovered the viruses of bacteria (Abedon, 2011). The bacteriophage era began when Felix d'Hérelle published a seminal publication that demonstrated “un bactériophage obligatoire” (Abedon, 2011) Soon after, microbiologists began to include the phages idea into their ideas. Novel therapies are needed to kill pathogenic bacteria. Phage therapy offers an alternative to the antibiotic treatment of bacteria. Even though bacteria can gradually resist phage, the resistance will not be as pronounced as the resistance to bacteria. Phage therapy is specific

Bacteriophage, or simply phage, are obligate intracellular viruses that invade bacterial cells and take over the cellular machinery to replicate their viral genome and create new viral progeny via lysis of the bacterial host cell (Buckling and Rainey 2002). Phage have been identified as the

Transduction is a method of transferring genes among bacteria via virus particles, which can be divided into two categories known as generalized and specialized transduction. During the lytic cycle, which quickly replicates phages and eventually releases them, ultimately killing the host cell, generalized transduction occurs. In generalized transduction, bacterial DNA is transferred from one cell to another by means of a bacteriophage. The phage first attaches to the bacterial host cell, and releases its nucleic acid in to the host cell, where host DNA is broken down. Phage protein coats (capsids) are formed in the cell, containing not only phage DNA, but mistakenly the bacterial host cells DNA as well. After the newly formed phages are released, the bacterial DNA

Bacteriophages (phages) are viruses that infect and multiply only in bacterial cells. They are not capable of infecting eukaryotic cells (human, animal, fungus, plants and insects). Phages are the natural predators of bacteria. Like all viruses, phages hijack the energy and cellular machinery provided by a host cell to replicate and make viral copies since they do not have their own metabolism. Endolysin enzyme encoded in the bacteriophage genome lyse the peptidoglycan of the bacterial cell wall during the phage lytic cycle, releasing dozens or hundreds of new phages (7). The cycle continues until there are no bacteria left to attack. Since phages only attack specific types of bacteria, they are unlikely to harm any human cells. In addition,

Generalized transduction is the transfer of any portion of the bacterial genome, while specialized transduction is the transfer of only specific portions of the bacterial genome. Unlike generalized transduction, specialized transduction is only carried out by temperate phages in the lysogenic cycle. When a phage infects a bacterial cell, phage DNA enters the cell and integrates into a specific site of the host chromosome. When the prophage is induced to excise, an error sometimes occurs, resulting in a phage genome containing some bacterial DNA and viral DNA. Due to the 5-10% of the bacterial DNA next to the integration site coupled with the viral DNA, some of the viral DNA is lost making the bacteriophage unable to reproduce. When the next host is infected, the bacterial genes will be transferred, leading to a merozygote, which we also see in generalized transduction by the transfer of bacterial genes to bacterium. Different phages that infect E. coli can accurately describe both types of

The increased use of antibiotics, development of resistance to these antibiotics and bacteriophage’s potential as a treatment model has forced the scientists to turn from antibiotic research to bacteriophages research. The use of bacteriophages in treating bacterial infections in humans as well as other species is termed as phage therapy.

As shown in the above results finding a phage is not guaranteed. Even when using multiple different soil samples and different protocol to isolate a phage there is still chance you won’t find a phage. There were no phages present within any of the plates there are a lot of reasons why this might be. The main reason is probably because of possible errors such as removing from the aseptic zone which would prevent contamination or having improper techniques for top agar pouring which could also screw the data. A lot of the soil samples collected were very dense and when we got our enrichment medium soil mixture back from the shakers we did notice that our soil samples had not separated as much as other groups. This could have caused contamination

Phage therapy is one novel area of research that should be explored in the treatment, prevention and control of these bacteria. This is due to the fact that it targets the specific bacteria and resistance to it is minimal. Bacteria such as Mycobacterium leprae, Streptococcus pneumonie and Mycobacterium tuberculosis over time have developed resistance to antibiotics. Mycobacterium leprae currently requires multidrug treatment due to the fact that it developed resistance to dapsone, which was an effective treatment. These bacteria have limited resistance to phage therapy hence making the intervention an effective treatment method. Even if these bacteria develop resistance to the phage, the resistance may be very easy to overcome as compared

Serial dilutions, also known as limiting dilution series, help to determine an estimation of how many phages/viruses are in a given culture (Zelterman et al., 2010). In this experiment we determined how many phages were in our given Salmonella typhimurium culture by doing three serial dilutions of an original phage suspension. This is important because it will allow us to get a rough estimation of how many phages where in our original sample and also provide us with knowledge on how to perform serial dilutions with phages which can be used to determine an unknown sample in the future. We expect that there will be less plaques on the last dilution than in the first plated dilution because the more diluted each test tube is the less phages are