Unadsorbed Phage Lab Report

# of plaques/(volume plated x reciprocal of the dilution factor of the dilution tube used)

Isolating and characterize a novel phage from the environment requires several steps and several frustrations. By isolating and investigating a phage found in the Pullman region can hopefully lead to a newly discovered phage that can help researchers discover more about the life cycle and process of phage infection. Some phage infection can be good due to infecting the bacteria that is not wanted or is harmful to the environment or humans. Within this lab, there were steps taken necessary to isolate a novel phage that was obtained from the surrounding Pullman area. This report reflects plaques being isolated but then stopped due to errors and loss of plates. The final touches and procedures were accomplished with a given DNA ladder that was

Bacteriophages were first discovered about 100 years ago by Frederick Twort and Felix D'Herelle. Bacteriophages or commonly known as “phages” are viruses that infect bacterial hosts. Bacteriophages come in multiple shapes and sizes. But a good amount of them are tailed viruses that contain double stranded DNA. The head of the bacteriophages has a protein shell which is attached to the tail. Some phages differ as some don’t have double stranded DNA they may be single stranded. Phages are incapable of reproducing on their own. In fact, they need a bacterial host to reproduce. Like any virus, bacteriophages are very fixed to discrete hosts. The host bacteria that we are using is called arthrobacter. Arthrobacter is a genus of bacteria found in soil (Pope et al 2016). Arthrobacter can help reduce hexavalent chromium which can cause some symptoms such as irritations to humans. Hexavalent chromium may also cause lung cancer if inhaled. The SEA PHAGE project was broken down into two parts. The first part which we are currently doing is isolating, purifying and amplifying the bacteria from our soil samples that we collected in the beginning of the year.

Phage display is a modern laboratory technique that is concerned with the study of not only protein-protein interactions but also protein-peptide and protein-DNA interactions through the use of a bacteriophage. Phage display uses a bacteriophage or a virus that infects bacteria to connect phenotype with genotype through the expression of a gene. This novel technique first starts by inserting a gene of interest, which codes for a protein of interest, into the coat protein gene of the virus, thereby causing the virus to express the gene both inside and outside in the form of a phenotype. The phages that display a particular gene can be compared or rather screened against other proteins, peptides, and DNA sequences to establish those above-mentioned interactions. The dynamic range of

The SEA Phage Project is a national project that keeps in account of different phage discoveries across the country. This research course for the two semester starts with digging up soil to find rare and similar phages (http://seaphages.org/). Started by the Howard Hughes Medical, it helps students get the experience of being in an advance lab session and also helps with advancing in their science career of their choice in genome annotation and bioinformatic analysis (http://seaphages.org/). The program requires a set of protocols used into completing this research. Its components are 1. Isolation, purification and amplification, 2. Bioinformatic and genomic features (Poxleitner, M, et al). The purpose of this research is to learn different lab techniques and experience a real- life lab work that is able to stay with students and use it for future references.

Next the tubes were placed in an ice bath, while obtaining a sterile loop to swipe a single colony of E.coli to put into the tube. After gently swiping a colony onto the loop, it was then spun in the +pGLO tube to get it to come off, returned to the ice bath. Next using a different sterile loop, it was swooped it in a container labeled pGLO plasmid DNA and again spun it ONLY into the tube with the solution labeled +pGLO to get it to come off. After about 10 minutes on ice the tubes were then placed into a 42ºC water bath for 50 seconds exactly, and immediately after placed them back into the ice bath. Finally, after 2 more minutes in the ice bath the tubes were separated into 4 containers. 250 ul of +pGLO solution was added to the containers containing +pGLO, LB broth, with ampicillin and +pGLO, LB broth, ampicillin, with arabinose. Also 250 ul of the –pGLO solution was added to the 2 containers containing LB broth, and ampicillin, with LB broth. Using a sterile loop for each plate the solutions were spread out gently and thoroughly on to the containers with agar. After the containers were incubated in 37ºC for at least 24 hours, the results were observed and disposed of (Weedman,

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

By the end of this experiment, the research objective had been achieved. Zassy was successfully characterized and analyzed using molecular cloning techniques. According to PhagesDB, there are 55 documented K1 bacteriophages. PhagesDB also shows that a majority of the bacteriophage found on Gonzaga University’s campus have been identified as being part of the K cluster. In addition, both Urkel and SamuelLPlaqson were found on Gonzaga’s campus.

The purpose of this study was to see whether E. Coli cells would engage in the pGLO plasmid and glow in the presence of four control environmental factors which are arabinose sugar, bacteria, the antibiotic ampicillin, LB nutrient broth and pGLO plasmid DNA. This was tested using four plates, all the plates had E. Coli cells and different environmental factors. The founding was that E. Coli will only fluoresce when bacteria, pGLO plasmid DNA, the antibiotic ampicillin, and LB nutrient broth are present. The result did not support the hypothesis because it stated that, E. coli cells that are exposed to the pGLO plasmid would engage in the plasmid and glow only if the arabinose sugar is present.

Bacterial transformation is the process of moving genes from a living thing to another with the help of a plasmid.The plasmid is able to help replicate the chromosomes by themselves; laboratories use these to aid in gene multiplication. Bacterial transformation is relevant in everyday lives due to the fact that almost all plasmids carry a bacterial origin of replication and an antibiotic resistance gene(“Addgene: Protocol - How to Do a Bacterial

Each batch of phage was used to infect a different culture of bacteria. After infection had taken place, each culture was whirled in a blender, removing any remaining phage and phage parts from the outside of the bacterial cells. Finally, the cultures were centrifuged, or spun at high speeds, to separate the bacteria from the phage debris.

14 Davis B, Waldor M (2003). "Filamentous phages linked to virulence of Vibrio cholerae". Curr Opin Microbiol. 6 (1): 35–42. doi:10.1016/S1369-5274(02)00005-X. PMID 12615217.

To start the experiment two small plastic tubes were labeled negative pGLO and positive pGLO as the first step in this experiment. A pipet was then used to move 250 microliters of calcium chloride into each tube and the tubes were iced. Bacteria was added using a new sterilized loop each time to the positive and negative pGLO tubes. The loop was twisted around in the tube to ensure that the bacteria was evenly mixed into the solutions and the tubes were iced once again. Another loop was dipped into the tube containing the plasmid and it was removed when there was a visible slight film of residue. The plasmid was dipped into the tube that was labeled positive for the pGLO gene. The two tubes labeled positive pGLO and negative pGLO were iced

In preparing for the bacterial transformation, DNA plasmid is introduced into the E. coli cells that will express newly acquired genes. Two tubes were used and labeled both as +pGLO and -pGLO. A solution of (CaCl2) was transferred 250 µl onto the two tubes. The tubes were placed on the ice. A sterile loop was then used to gather a single colony of bacteria from a starter plate. Now, that both tubes contain bacteria they were placed on the ice for 10 minutes. Four agar plates were labeled as: +pGLO LB/amp, +pGLO LB/amp/ara, +pGLO LB, -PGLO LB/amp. Heat shock was used to transfer both the +pGLO and -pGLO, at exactly 42°C. Time was observed for 50 seconds and quickly return the tubes to the ice for another 2 minutes. As the tubes, cold down they

Abstract:Conjugation is a natural occurring process that involves the transfer of DNA from one cell into another through a physical connection between the cells. In the following experiment, two strains of Escherichia coli bacterial cells (donor F'lac+strs and recipient F-lac-strr) underwent conjugation to produce a transconjugant strain (F'lac+strr). MAC plates and streptomycin were utilized to determine if conjugation had occurred. When plated, the donor colonies appeared red and the recipient colonies appeared white. The transconjugant plates showed red and white colonies. Using alkaline lysis miniprep, a DNA plasmid was isolated from the donor and transconjugant strains and FIGE electrophoresis was used to determine the size of the