Phage From The Environment

The experimental part of the lab consists of setting up the materials needed. A sample of E.coli and a solution of calcium chloride are first obtained and placed in different test tubes. 630µL of Calcium Chloride (CaCl2) are then removed from the test tube and inserted into the test tube containing E.coli cells (Alberte et al., 2012). The newly formed substance of Calcium Chloride and E.coli is then mixed and incubated in ice for 10 minutes, making the cells more competent. Two test tubes are obtained and labeled; the first test tube is labeled with pUC18 and the second one with “Lux” to represent the plasmids being used. These two test tubes are then incubated in ice. 3µl of the set plasmid are added to each of the two test tubes. The test tubes are tapped to guarantee the cells are well

6 petri dishes were labeled, 3 petri dishes P 105 B, P 106 B, P 107 B, and the other 3 petri dishes P 10 K, P 102 K, P 103 K and they were set aside. A series of dilutions for the 1x bacteriophage T4 rII

Placed it to the shaking incubator at 37˚C to prewarm. Heat shocked the cells for 60 seconds in 42°C water bath. Added 950 µl of warm (37°C) SOC medium and incubated with shaking (160-225rpm) for 50 minutes at 37˚C. Used two LB plates. Plate 100 µl of each transformation on a separate LB agar plate containing carbenicillin which is the antibiotic, IPTG to induce expression of lacZ and X-gal a substrate for β-galactosidase. The plastic L-spreader was used to distribute the bacteria.

This project is all about isolating bacteriophage in soil. They come in different sizes and shapes, each to their own unique look. Phages have a protective protein head that contains DNA and a hollow tube tails (http://phages.org/bacteriophage/). Since bacteriophage cannot reproduce and replicate themselves, they need a host to do the work for

The validity of my experiment is invalid as looking back on how I performed the experiment, I would say that I made one mistakes that I believe would have affected the overall result. This mistake is not waiting long enough for the water to dry on the sprayed agar plates. By not waiting long enough, the water would still carry bacteria. Meaning that even though it wouldn’t be there when the water dried, it was still there when I ran the swab over the top of it.

Three of the circles were labelled with the number 1, three were labelled 2 and three were labelled 3, leaving three blank. The swab was removed from the packet and quickly placed into the bottle of e. coli bacteria then quickly being removed with no dripping chemicals. The e. coli bottle was then closed while the swab applied the e. coli onto the base of the agar plate making an ‘s’ shape pattern, and then turned 90 degrees and was repeated. After this was completed the lid was placed on that agar plate while one of the small circles labelled number one was dipped into a small amount of detergent. The lid was removed from the agar plate and the paper was placed onto the top left corner of the agar plate using tweezers. Once it was flat on the base with the number one facing up, the lid was again put on the agar plate to prevent bacteria from the air landing on the surface. Using a new, clean pair of tweezers a piece of paper labelled with the number two was dipped into hand sanitiser, the lid was removed and the paper was placed flat on the top right corner of the base of the plate with the lid then being replaced on the plate. With another pair of

The purpose of the following experiment was to isolate and analyze a novel phage from the environment [3]. Initially, the phage was extracted from the environment through an enrichment procedure [3]. The first enrichment was successful producing a two cloudy plaques which were theorized to be temperate phage. It was theorized to be a temperate phage due to the cloudy appearance of the phage plaque. The two streaking procedures that followed did not result in any visible phage. This could have been due to two possibilities, either a flawed lab technique or the phage that had originally been enriched had died off. In order to determine whether the phage had died an additional enrichment was conducted. The second enrichment resulted in no visible phage plaques.

See Figure 7. Figure 8, Control After Incubation, shows no bacterial growth for all agar plates shown. This is a good thing, means the control was not contaminated. See Figure

Experimental Method: The experimental design was sourced from the lab course manual: Setting-up RNAi With a light microscope, two live worms were located on the control plate. 180 μl of M9 buffer was mixed with 20 μl of 1M IPTG. The entire 200 μl was pipetted into the plate covering the bacteria. The plate sat for ten minutes until the liquid was completely absorbed. The worms were put under the microscope to ensure they were still alive.

Turn the starch agar plates upside down and draw two lines with the sharpie on each, dividing each petri dish into four separate sections. Label the sections 1A, 1B, 1C, and 1D on the first petri dish, 2A, 2B, 2C, and 2D on the second, and so on. 2. Pass forceps through the Bunsen burner flame; let them cool for a little bit, then use them to pick up one of the paper discs. Open the culture of Bacillus subtilis, flame the neck of the bottle, and dip the disc in the broth.

The first task we had to perform in this lab was diluting an Escherichia coli culture and compare it to the

The T-Even bacteriophage is a virus that attacks E. Coli bacteria. The basic function for all viruses is alike. Viruses use this function to hijack cells and inject their own genetic information to reproduce themselves. One cell alone can reproduce hundreds of viruses. This particular virus is good for you, because it kills off the E Coli. infection, which means this is a medical virus that can be used to treat illnesses.

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

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

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.