Characterizing Bacteriophages Found in Water Sample

Writer: Megan Troxel

Lab Partners: Mariah Blecher, Keith Frey, Tyser Lyfoung, Sofia Rahmanzai

Introduction:

The goal of the overall experiment is to figure out the genome sequence of the virus

found in the water sample. The specific goal in the part of the lab is to figure out different

characterizations of the virus such as virus type, size, concentration and other factors about the

virus in the water sample. Figuring out these characterizations will help further our knowledge

on the virus found in this water sample.

First we need to isolate our viral genome. Nucleic acid extraction is done to get the

isolated viral genome. Extraction is used to digest contaminating cellular nucleic acid before the

virus genome is extracted from the viral capsid. Digestion of these capsid proteins are done using

phenol and phenol chloroform followed by ethanol precipitation to recover viral nucleic acids

and genome. After this process, characterization of the genome can be taken with the Nanodrop

2000. Only a small drop is needed to use the Nanodrop 2000. It takes the concentration of the

nucleic acid in the sample by using UV light. The software will do all the calculations to

determine the concentration and the purity.

Restriction of the viral DNA is then done by performing an agarose gel electrophoresis.

Restriction allows certain DNA sequences to be recognized and then make scissions in the

DNA. In this experiment this screening is done on unknown viral genome. These are done in

diagnostic restriction digest. This will be done by agarose gel electrophoresis. This method is

used to separate a complex mixture of molecules characterized by their rate of movement

through the matrix by magnetic fields. Molecules react with the charge that allows it to be pulled

through the gel. Smaller molecules will also move faster than the larger ones, so these bands can

actually be read. This is done in many different lab techniques to characterize size of DNA and

proteins. The gel is prepared and set. A comb is used to create wells to where the sample can be

put into. Positive and negative electrodes are attached, one on each end to create the electrical

current through the gel to move the sample at a set voltage.

Another method to characterize viruses can be done by using electron microscopy. This is

a very amazing technique and was done at UNMC. The sample was negatively stained and then

scanned through to find viruses. This gives us insight on how the virus looks and what shape and

type the virus may be. This is done by staining the virus and shining a beam of light through it to

get clear images under the microscope. Then measurements can be taken and pictures taken as

well.

Methods:

Nucleic Acid Extraction from Virion:

Remove two aliquots of 0.5 ml of viral stock into separate eppendoft tubes. Then, 5 l of

DNase I and 5 l of RNase A was added. This was incubated for 30 minutes at room

temperature. Then after incubation, 5 l of proteinase K and 25 l of SDS was added to each

tube and incubated for 1 hour at 56C.

Next is phenol extraction and ethanol precipitation after incubation of an hour. This is all

done in the hood due to how dangerous phenol is. Gloves and eye protection were to be worn as

well. To this mixture, 500 l of phenol was added, vortexed 30 seconds and then separated into

phases by centrifugation for 2 minutes. After 2 minutes, remove the upper aqueous phase into a
new microfuge tube. Then add 500 l of phenol-chloroform, vortex for 30 seconds and then

centrifugation for 2 minutes. Again, transfer the upper aqueous phase into a new tube. This time,

add 500 l of chloroform, vortex for 30 seconds and centrifuge for 2 minutes. Again transfer the

upper aqueous portion.

Next is the ethanol precipitation. Add 50 l of 3 M NaOAc with a pH of 7.0 and 1 ml of

95% ethanol to this tube. Mix by inverting and observe the solution. Store overnight at -20C. To

recover the nucleic acid, centrifuge in a microfuge for 10 minutes at 4C. Discard the supernatant

and was the pellet with 600 l of cold 70% ethanol without mixing. Centrifuge for 5 minutes in a

microfuge. Discard the supernatant, dry the pellet for 2 minutes and resuspend the pellet in 50 l

of TE pH 8.0. Place a drop on the nanodrop to determine the concentration.

*Note: this was done twice; once on 2/22/17 and 3/8/17. Readings of the Nano 2000 were

taken 3/8/17

Gel Electrophoresis

TAE Buffer

50X TAE stock was made first.

50X TAE Stock
2M Tris 24.2 g/100 ml
Acetic acid 5.7 ml/100 ml
50 mM EDTA 10 ml of 0.5M stock, pH 8

- To make 10X TAE buffer with 5 ug/ml EthBr, dilute 10 ml of 50X to 50 ml then 25 l

of 10 mg/ml EthBr was added.

- To make 1X TAE with 1 ug/ml EthBr, dilute 20 ml of 50X to 1 liter then 100 l of 10

mg/ml EthBr stock was added. This was the gel running buffer.

Preparation of Agarose Gel:

 We made a 30 ml gel. First, 0.5 g of agarose was placed into 25 ml of dH2O in a 125ml

flask. The agarose was completely melted in the microwave and boiled to dissolve the agarose.

More water was added to replace the water that was boiled away. Then 5 ml of 10X TAE buffer

containing 5ug/ml Ethidium Bromide was added. The solution was cooled to less than 60C then

poured into the gel tray. A comb was placed into the gel to form the wells and the gel then

solidified.

To load the gel, a total of 10 l was loaded. Using the concentration determined from the

Nano 2000, we wanted 1 g of the nucleic acid so we determined to use 3.5 l of our nucleic

acid, 2 l of loading dye and 4.5 l of water. The specs on the machine was the following: time-

59, volts- 69 and mA- 49. The positive end of the electrode was orientated away from the wells.

Negative Staining:

This was done at UNMC in their lab. A 30 l drop was moved onto parafilm.

With a Formvar and carbon coated copper grid is floated with a coated surface on the drop of

suspension for 1 minute then the excess was wiped away and air dried for 30 seconds. Then 30

l of negative staining solution was put onto the film. The grid with sample absorbed to the

surface is floated on the drop of negative stain for 2 minutes and again excess was wiped off and

hair tired for 60 minutes. Then the grid is examined by the transmission electron microscope.

Picture were taken of the found virus.

Results:

Nucleic Acid Extraction

Strands of visible DNA wasnt noticeable until a week later when it was checked. There

were white, stringy looking particles in a slightly cloudy clear solution.

Nanodrop 2000

Readings for the Nanodrop were recorded March 8th, 2017 using the nucleic acid

extraction. In figure 1 below, there is a slope at 260 nm which shows that there is nucleic acids in

our solution. The concentration was read at 281.3 ng/l. The ratio of nucleic acids is 1.77.

Figure 1: Nanodrop 2000 result with 260 nm at the highest peak.

Gel Electrophoresis

The agarose gel electrophoresis was processed on March 8th, 2017. Looking at lane 4,

there is a very faint line towards the top. Perhaps the nucleic acid didnt pick up enough stain to

be shown on the gel.

1 2 3 4 5 6 7 8
 + end
Figure 2: Agarose gel. Lane 4 is group 3 and there is a very faint line towards the top.

Electron Microscopy

According to the pictures and measurements, the head length is 78.26-91.48 nm long, the

width of the head is 68.05-78.18 nm wide, tail length is 123.9 nm long and tail width is 20.76 nm

wide. The average head length is about 75 nm and 78 nm wide. Photographs are at 110,000 times

magnification. Pictures are shown in picture 1-4.

Picture 1 and 2: these show the length and width of the head

Picture 3: appears to be infecting a particle. Picture 4: measurements of head and also of the

head.

Discussion:

According to all our observations, Small Private B is a bacteriophage. After looking at the

microscopy, it appears to a bacteriophage with its distinctive head and tail shape.
Later we will do a one-step growth curve for our virus. Still want to keep trying SDS-PAGE. We

will need a higher concentration.