MCB 3020 - Chapter 19-21 Quiz
Describe the term "obligate intracellular parasite":
A virus that can reproduce only within a living cell. The term obligate refers to something that must behave in a specified manner and intracellular means located within the cell.
When a diluted aliquot of a virus sample is added to bacterial culture of a suitable host:
An infectious cycle will begin if conditions are correct.
Fourth step of virus infection:
Assembly - the phage chromosomes are packaged inside capsids, phage particles spontaneously assemble.
First step of virus infection:
Attachment - the phage attaches to the exterior of the cell on a receptor molecule and binds with it; geometric/chemical fit.
What are the steps for viral replication:
Attachment, penetration, protein synthesis, assembly, and release.
Which strain is the recepient?
BS phe-, auxotroph
Which strain is the donor?
BS wild type, prototroph
How can selection for cells that have been transformed with pGLO DNA be accomplished?
By growth on ampicillin plates, beta-lactamase destroys ampicillin. Appear white on plates not containing arabinose and fluorescent green under UV light when arabinose is included in the nutrient agar.
How do you select for transformants?
By using the GMS plate, no growth would normally be seen but because of WT DNA there will be some growth with a few colonies.
Artificial Transformation:
Carried out in the laboratory by a variety of artificial means that make naturally incompetent bacterial, such as E. coli, competent by rendering the bacterial cell membrane more permeable to DNA.
Calcium Chloride solution:
Contains 100 mM Calcium Chloride and 15% glycerol. Glycerol is a cryoprotectant. The addition of glycerol to the calcium chloride solution allows you to freeze the competent cells before the addition of the plasmid.
Natural transformation:
DNA comes from a donor bacterium (for example, after cell lysis) and naturally competent bacterial cell, incorporates this DNA into its chromosome. Occurs with Streptococcus and Bacillus.
Fred Griffith:
Discovered transformation in the late 1920s, studied bacterium Streptococcus pneumoniae which occurred as a smooth (S) colony - highly effective and rough (R) colony - harmless. In an experiment with mice, heat-killed S cells cells transformed some of the living R cells into harmful S type cells.
Second method to produce competency in naturally incompetent bacteria and to transform these bacteria artificially:
Electroporation, cells are subjected to high voltage electric impulses that destabilize the cell membrane. Results in increased permeability and enabling DNA to pass into the cells.
True or false, all viruses use dsDNA as their genetic material:
False
Which plate is the transformation?
GMS + phe- + WT DNA = few colonies
Which plate is the negative control?
GMS + phe- = no growth
Naturally competent bacterial cell:
One that is able to take up DNA and be transformed, doesn't have to be forced.
How do you tell the difference between a lysogenic or lytic infection on a lawn of bacteria:
In the lytic cycle, the bacteriophage kills the bacteria. While in lysogenic cycle, the bacteria survive. So more plaques would be seen on the lawn of the lytic infection than the lysogenic infection.
Which plate is the positive control?
LB + phe- = growth
Prototrophs:
Most bacteria which are capable of synthesizing all of the required amino acids needed for survival.
Virus:
Obligate intracellular parasites that are incapable of reproduction outside of a host organism.
Fifth step of virus infection:
Release - the phage proteins lyse host cell; new phage particles are released.
Example of calculation for titer:
Serial dilute a virus using 100 microliters of virus into 900 microliters of LB, dilution factor is 1:10. 100 microliters of each dilution to plates, 80 plaques visible on the fifth plate. Using titer formula, pfu/ml: 80 pfu/0.1ml x 10^-5 = 8 x 10^7 pfu/ml.
How can you switch on the gene for GFP in transformed cells?
Simply by adding the sugar arabinose to the cell's nutrient medium.
Auxotrophs:
Some strains of bacteria that have genetic mutation that prevent them from synthesizing one or more of the required amino acids.
Transformation:
The transfer of genetic material from one bacterium to another, whereby one bacterial cell takes up the naked DNA from the environment.
First method to produce competency in naturally incompetent bacteria and to transform these bacteria artificially:
Treatment of bacterial cells with ice-cold calcium chloride, which enables the cells to uptake DNA from their environment.
"T-even" bacteriophage:
lytic infection, E. coli strain B.
Plaque assay:
method of determining the concentration of viral particles in a sample. Developed to count and measure infectivity of bacteriophages.
It only takes ______ virus to infect a bacterial cell and initiate a lytic infection
one
What is the plasmid used for the transformation of E. coli?
pGLO plasmid, contains several reporter genes. Most notably for the Green Fluorescent Protein - GFP and the beta-lactamase antibiotic resistance gene - bla.
Plasmid cloning vectors:
plasmids that are used for transformation experiments and, therefore, have been genetically engineered.
If the concentration of the virus is too high:
possibly all of the bacteria will be affected.
Protein coat:
the coiled or polyhedral structure, composed of proteins, that encloses the nucleic acid of a virus.
Capsid:
the protein shell of a virus and encloses the genetic material of the virus.
If the concentration of the virus is too low:
there may not be any visible plaques.
Viruses don't need to maintain a vast amount of genetic information because:
they reproduce by using the host cell's synthetic machinery and materials
The concentration of virus particles, or titer, in the virus sample:
titer - pfu/ml = number of plaqus/quantity of virus sample x virus dilution
Plaque:
a clear area on an otherwise opaque field of bacteria that indicates the inhibition of the bacterial cells by either a virus or an antibiotic.
The infection observed as a plaque is the result of:
a single virus particle infecting a bacterium and the resulting lytic cycle.
Bacteriophage/"phage":
bacterial virus that invades bacterial cells and, in the case of lytic phages, disrupts bacterial metabolism and causes the bacterium to lyse.
Some viruses are specific to bacterial hosts, so they are classified as:
bacteriophages or just the shorten term "phage".
Tail sheath:
contracts to perforate the cell wall with the viral pin
Different families of virus use:
dsDNA, single stranded RNA - ssRNA -, ssDNA, or dsRNA.
The concentration of the bacterial host should be sufficient enough to:
form a "lawn" of bacteria on the agar surface.
Viruses have their genetic material stored in the:
head/capsid
Temperate viruses:
incorporate their genetic material into the host cell's genome in the lysogenic pathway - stays dormant until favorable conditions for virus to be released.
In order for an auxotrophic strain of bacteria to survive:
it must be supplemented with the missing amino acids. Normal enriched media contain all the required amino acids, but minimal media does not and, this, inhibits the growth of auxotrophic strains.
Viruses do not ____________ in any type of media
"grow"
Second step of virus infection:
Penetration - the phage penetrates the cell wall/membrane either by cellular phagocytosis or direct injection and injects its chromosome into the host cell.
Third step of virus infection - part 2
Protein synthesis - lysogenic infection: phage proteins synthesized in host cell, phage chromosome circularized by phage protein, phage is dormant and once broken the lytic infection is initiated, phage components synthesized.
Third step of virus infection - part 1
Protein synthesis - lytic infection: phage proteins synthesized in host cell, host chromosome degraded by phage proteins, phage components synthesized.
Tail fibers:
Proteins on fibers that are specific for receptors on the surface of a bacterium.
Plasmid:
Used to transform bacterial cells. Small circular pieces of extrachromosomal, double-stranded DNA.
