Microbiology Lab Chpt 19-22
Why is 10% SDS (sodium dodecyl sulfate) added to the wild type cells in experiment 19?
"Solubilizes" the cell membrane which leads to the cell lysis of the bacterial cells
What are the number of fragments after digestion in circular DNA?
# of restriction sites
What are the number of fragments after digestion in linear DNA?
# of restriction sites + 1
Restriction fragments
- A set of reproducible DNA segments that result from the cutting of DNA by a restriction enzyme - Have single-stranded overhangs
What is single nucleotide polymorphism (SNP)?
- A single nucleotide variation in DNA sequence - A/T/C/G differs between members of a species or paired chromosomes.
What are XL1 Blue II cells?
- An E. coli strain that has a mutated recA gene (recA-) - Cannot repair damage to DNA caused by UV radiation and other environmental agents
How will transformed cells appear in the presence and absence of arabinose?
- Appear white (wild-type phenotype) in absence of arabinose - Appear fluorescent green under UV light in the presence of arabinose
Prototroph
- Bacteria capable of synthesizing all of the required amino acids needed for survival - Grows faster on rich media than minimal media
Auxotroph
- Bacteria with genetic mutations in essential metabolic pathways that prevent them from synthesizing one or more of the required amino acids - Can only grow on rich medium, not minimal medium
Beta-lactamase antibiotic resistance gene (bla)
- Beta-lactamase destroys ampicillin - Allows for selection of cells that have been transformed with pGLO DNA by plating on ampicillin plates
How does ice-cold CaCl2 treatment work?
- CaCl2 at a concentration of 100mM makes the cell membrane more permeable - Allows foreign DNA from the environment to pass through the cell membrane and into the cell
What is electroporation?
- Cells are subjected to high voltage electric impulses that destabilize the cell membrane, resulting in increased permeability - Enables DNA to pass into the cells
Why are XL1 Blue II cells used in experiment 20?
- Cells do not have RecA gene, so it is easier to penetrate the cell membrane and allow the DNA to enter the cell - Cells cannot repair damage to DNA during ice-cold CaCl2 treatment and heat shock
Green fluorescent protein (GFP)
- Comes from bioluminescent jellyfish - Generates bioluminescence under UV light
Titer
- Concentration of virus particles in virus sample - in pfu/ml of plaque forming units
Tail fibers
- Consist of proteins that are specific for receptors on the surface of a bacterium - Used for attachment to the host cell
Tail sheath
- Contracts to perforate the cell wall with the viral pin - Nucleic acids are pumped from the capsid, through the tail sheath and pin, and into the protoplasm of the bacterial host
What are the benefits of DNA fingerprinting?
- Convicting felons - Exonerating the innocent - Establishing maternity and paternity - Proving family relationships
Restriction fragment length polymorphisms (RFLPs)
- Differences in the restriction sites on homologous chromosomes that result in different restriction fragment patterns - Loss of a site will result in the appearance of a larger fragment and the disappearance of two smaller fragments - Formation of a site will result in the disappearance of a larger fragment and the appearance of two smaller fragments
Polymorphism
- For every 1,000 nucleotides that we inherit, there is 1 site of variation in the population - May change the length of DNA fragments produced by restriction enzymes by either creating or destroying restriction enzyme recognition sites
What is the setback of using DNA in DNA fingerprinting?
- Genomic human DNA is around 3 billion base pairs - Genomic bacterial DNA is over 4 million base pairs - Too long so when cut with a restriction enzyme and the fragments are separated by gel electrophoresis, no clear restriction pattern can be seen - Only a smear of DNA representing fragments of just about every possible size is visible
Why is sample buffer added to the lambda phage DNA sample after treatment and digestion with restriction enzyme in experiment 22?
- Glycerol, increases the density of the sample - Bromophenol blue is a tracking dye, which is used as a visual aid
Lysogenic pathway
- Initiated by temperate viruses - Incorporate their genetic material into the host cell's genome - Host cell reproduces and produces daughter cells that contain the copies of viral genetic material - When conditions are favorable for the virus to be released, the viral nucleic acids will direct the cellular metabolism to produce new virus particles and switch to the lytic cycle
Lytic pathway
- Initiated by virulent viruses - Virus lyses host cell to release new viral particles and spread genetic material
Why is lambda phage DNA used in experiment 22?
- Linear DNA - Approximately 50 kbp
Why is resuspension buffer added to the wild type cells in experiment 19?
- Lysozyme hydrolyzes the peptidoglycan in the bacterial cell walls and lyses the cell - RNase degrades DNA and digests the RNA that is released by the lysis of the cells
What does a higher agarose % in the gel do?
- More restrictive - Separates small fragments of DNA more easily
Virus
- Obligate intracellular parasites that are incapable of reproduction outside of a host organism - Cannot "grow" in any type of media - Consist of a protein coat containing nucleic acids
What percentage of human DNA is identical?
- Over 99% of all 3 billion nucleotides in human DNA are identical among all individuals
Plasmid cloning vectors
- Plasmids that are used for transformation experiments - Genetically engineered - Contain an antibiotic resistance gene and a region in which exogenous DNA fragments can be inserted
Restriction enzymes
- Produced by bacteria as a defense mechanism against phage infection - Recognize 4-9 bp sequences, called restriction sites, and then cleave both DNA strands at these sites - Also called restriction endonucleases because they cleave DNA within the molecule
AraC gene
- Regulates the expression of GFP in the presence of the sugar arabinose - GFP can be switched on in transformed cells by adding arabinose to the cell's nutrient medium
R form of S. pneumoniae
- Rough colony - Harmless
Plasmid
- Small, circular pieces of extrachromosomal, double-stranded DNA that can be found in a variety of different bacteria species - Replicate independently of the bacterial chromosome - Range in size from a few thousand base pairs (bp) to more than 100 kilo base pairs (kb)
S form of S. pneumoniae
- Smooth colony - Highly infective
Agarose
- Solidifying agent - Forms a matrix within the gel that controls the migration of the restriction fragments - No charge
How does agarose gel electrophoresis work?
- The digested DNA sample is placed into wells near one end of a slab of agarose gel - A current is applied and travels to the end with the cathode (-) near the wells containing the DNA and the anode (+) on the opposite end - The phosphate group of the DNA molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode - The charge is cumulative, so longer molecules have greater negative charge - The smaller fragments travel easier and faster through the agarose than the larger fragment - All fragments of the same size travel together through the gel due to having the same size, charge, and length of base pairs
What is the purpose of bromophenol blue in the sample buffer in experiment 22?
- Tracking dye that is charged, so it travels slightly faster than the smallest fragments - When the dye nears the end of the gel, it indicates the current must be removed and the electrophoresis operation stopped
TAE buffer
- Used to conduct current both in the gel and the surrounding buffer - Contains Tris Acetate and EDTA
Describe the steps for viral replication.
- Viruses infect host cells by attaching to the exterior of the cell - Viruses then penetrate the cell wall/membrane either by cellular phagocytosis or direct injection - Viruses then release the viral genetic material into the cell, where viral nucleic acids direct the cellular metabolism to produce new viral proteins (protein synthesis) - New viral proteins and new copies of the viral nucleic acids are assembled within the host cell - The host cell is lysed and and the new virus particles are released
Bacteriophage
- Viruses that are specific to bacterial hosts - Shortened term --> "phage"
What kind of genetic information can viruses have?
- dsDNA - ssRNA - ssDNA - dsRNA
You need to make a 0.7% agarose gel with 40 ml of 1X TAE. How much agarose is needed?
0.7 g/100 ml = grams/ 40 ml 0.28 grams
What are the steps for viral replication?
1. Attachment 2. Penetration 3. Protein synthesis 4. Assembly 5. Release
What 2 properties of ethidium bromide make it valuable for visualizing DNA?
1. Binds to DNA and forms EtBr-DNA complex (POWERFUL CARCINOGEN) 2. It is a fluorescent dye
What structures do T-even bacteriophages consist of?
1. Capsid 2. Tail sheath 3. Tail fibers 4. Pin
What 2 strains of B. subtilis are used in experiment 19?
1. Donor strain wild type (prototroph) 2. Recipient strain phenylalanine auxotroph (phe-)
What 3 plates are used in experiment 19 and how are they labeled?
1. GMS plate A labeled "phe- cells and wild type DNA" 2. GMS plate B labeled "phe-cells" 3. LB agar plate C labeled "phe-cells"
What does sample buffer consist of in experiment 22?
1. Glycerol 2. Bromophenol blue
What does the pGLO plasmid contain?
1. Green fluorescent protein (GFP) 2. Beta-lactamase antibiotic resistance gene (bla) 3. AraC gene 4. Ori- origin of replication
What are 2 methods to artificially transform bacteria?
1. Ice-cold calcium chloride (CaCl2) treatment (+heat) 2. Electroporation
What does resuspension buffer consist of?
1. Lysozyme 2. RNase
What are the 2 pathways of viral reproduction?
1. Lytic pathway 2. Lysogenic pathway
Two kinds of transformation:
1. Natural transformation 2. Artifical transformation
What kind of growth will you expect to see on all 3 plates in experiment 19?
1. Plate A- small, isolated colonies 2. Plate B- no growth (negative control) 3. Plate C- lots of growth/confluent lawn (positive control)
What 2 forms of Streptococcus pneumoniae were used in Fred Griffith's experiment?
1. S form 2. R form
What is agarose gel made of?
1. TAE buffer 2. Agarose 3. Ethidium bromide (EtBr)
Name and describe the function of two of the reporter genes on the pGLO plasmid.
1. The bla gene encodes for the synthesis of beta-lactamase which degrades the antibiotic ampicillin. Bacteria with the bla gene can grow on medium that contain ampicillin. 2. AraC gene encodes for the activation of GFP in the presence of the sugar arabinose. Therefore, bacterial cells with AraC gene will be fluorescent under UV light when there is arabinose present in the medium.
What 2 different strains of B. subtilis are used in Experiment 19?
1. Wild type (prototroph)- DONOR STRAIN 2. Phenylalanine (auxotroph)- RECIPIENT STRAIN
You need 3.5 L of 50 mM NaCl from your 20X stock solution. How you accomplish this?
20X stock = 175 ml Pure Water = 3325 ml 3.5 L = 3500 ml C1V1 = C2V2 (20X)(V1) = (1X)(3500 ml) V1 = 175 ml 3500 ml - 175 ml = 3325 ml
Jax Teller needs to make 500 mL of a 100X stock solution of TAE buffer. The final working concentration of TAE buffer is 40 mM Tris and 2 mM EDTA. The M.W. for Tris is 121.4 g and the M.W. for EDTA is 372.24 g. How would he accomplish this? Show your work.
500 mL = 0.5 L Tris --> 40 mM = 0.04 mol/L EDTA --> 2 mM = 0.002 mol/L Tris M.W. --> 121.4 g EDTA M.W. --> 372.24 g Tris = (0.5 L)(121.4 g)(0.04 mol/L)(100) = 242.28 g EDTA = (0.5 L)(372.24 g)(0.002 mol/L)(100) = 37.224 g 500 mL of H2O
How many virus particles will produce a plaque?
A single virus particle infecting a bacterium!
You spread 100 microliters of B. subtilis phe- of an overnight culture onto a GMS (glucose minimal salt) plate and added DNA from B. subtilis met-. What result (kind of growth) did you expect on this plate? Discuss the results from the previous flashcard with this result.
B. subtilis phe- is a naturally competent bacterial cell and would be able to undergo natural transformation as a receiver when introduced to the donor bacterium, B. subtilis met-. When B. subtilis phe- cells and B. subtilis met- cells combine, B. subtilis phe- cells transform and are now able to grow on minimal medium, such as GMS plates. Not all B. subtilis phe- cells will undergo transformation, therefore, I expect to see larger amounts of colony growth on the GMS plates when compared to the GMS control from the previous flashcard, but a smaller amount of colony growth when compared to the LB plates from the previous flashcard.
You spread 100 microliters of B. subtilis phe- of an overnight culture onto an LB plate and onto a GMS (glucose minimal salt) plate. What results (kind of growth) did you expect? What was the purpose of these two plates in this experiment?
B. subtilis phe- is an auxotrophic microoorganism, which means that it contains one or more mutations in essential metabolic pathways. Auxotrophic bacteria will grow on rich medium, but not on minimal medium. I would expect to see growth of larger colonies of B. subtilis phe- on the LB plate and no growth on the GMS plate. The LB plate and GMS plate are control plates that are used as a comparison when transforming the B. subtilis phe- using the prototrophic B. subtilis cells.
What is the bacteria of interest in experiment 19?
Bacillus subtilis
How would he prepare 200 mL of a 1X TAE buffer from his 100X stock solution from the previous flashcard?
C1V1 = C2V2 (100X)(V1) = (1X)(200 mL) V1 = 2 mL of 100X stock solution 200 mL - 2 mL = 198 mL of H2O
Viruses have their genetic material stored in the _________________.
Capsid
Artificial transformation
Carried out in the laboratory, naturally incompetent bacteria, such as E. coli, are made competent by making the bacterial cell membrane more permeable to DNA
Capsid
Contains the nucleic material (chromosome)
What is the viscous precipitate that forms with the addition of ice cold EtOH in experiment 19?
DNA
Natural transformation
DNA from a donor bacterium is taken up by a naturally competent bacterial cell- one able to take up DNA and be transformed- and incorporated into the chromosome
Why is proteinase added to the wild type cells in experiment 19?
Denatures most of the proteins in the cells which helps with the separation of the proteins from the DNA
Why is NaCl added to the wild type cells after incubation? in experiment 19?
Denatures the RNase and makes the DNA less hydrophilic by binding the negatively charged phosphate groups of the DNA with the Na+ ions
True or false, "All viruses use dsDNA as their genetic material".
False- Different families of viruses may use dsDNA, ssDNA, dsRNA, or ssRNA as their genetic material
You load one sample of undigested lambda phage DNA and one sample of KpnI digested lambda phage DNA on a 0.7% gel and on a 1% gel. Digestion of lambda phage DNA with KpnI gives you 3 fragments: 1503 bp, 17.1 kbp, and 29.9 kbp. What would you see on a UV transilluminator?
For the first gel with undigested lambda phage DNA, I would see only one large band, which moves very slowly from the loading site. Therefore, a single band will be visible at the top of the gel. For the second gel with KpnI digested lambda phage DNA, I would see 3 bands in the gel. The smaller fragments move the farthest through the gel, therefore, the 1503 bp fragment will be visible at the bottom of the gel, the 17.1 kbp fragment will be visible in the middle of the gel, and the 29.9 kbp fragment will be visible at the top of the gel, closest to the loading site.
Why did the mice in Fred Griffith's experiment die?
Heat-killed S cells transformed some of the R cells into virulent S type cells
Ethidium bromide (EtBr)
Helps to visualize DNA
During an artificial transformation experiment you accidently use an Eppendorf tube with E. coli wild type cells. You continue with the experiment and pipette 100 microliters of the cells from the "+pGLO" tube onto an LB agar plate and an LB/amp/ara plate. After incubation, you see a confluent lawn on the LB agar plate and no growth on the LB/amp/ara. Was this expected. Why or why not?
I did expect to see a confluent lawn on the LB agar plate and no growth on the LB/amp/ara plate because E. coli wild type cells were used instead of XL1 Blue II cells. XL1 Blue II cells have a mutated recA gene, which is responsible for the regulation of bacterial recombination of chromosomal DNA and the expression of various mechanisms, which repair damage to DNA caused by UV radiation and other environmental agents. The wild type cells are able to perform bacterial recombination and repair damage to DNA, therefore, they are not able to undergo transformation. Without transformation, the wild type cells do not acquire the bla gene, so the cells cannot survive on medium that contains ampicillin.
Assume that you plated your +pGLO culture on an LB agar plate. What result would you expect?
I would expect to see a confluent lawn when plating +pGLO culture on an LB agar plate because there is no ampicillin present. When ampicillin is present, there will be smaller, isolate colonies. With no antibiotic, the bacterial cell growth is unaffected. E. coli is a prototroph, so it grows best and fastest on LB agar, which is a rich medium. In addition, the lawn of bacteria will appear white and not fluoresce under UV light because there is no arabinose present in the medium.
What would happen if there was no bromophenol blue in the sample buffer in experiment 22?
If the current were continued, the DNA would continue migrating and end up off the gel and lost in the TAE buffer
Why should the water bath temperature not go over 50 degrees Celsius in experiment 21?
If the temperature is too high, the bacteria could be affected and die
How do you tell the difference between a lysogenic or lytic infection on a lawn of bacteria?
Lytic infection is initiated by virulent viruses and results in the lysis and cell death of the host cell to release new viral particles and spread genetic material. With lytic infection, there will be areas of the bacterial lawn where remnant of the dead, lysed bacterial cells will appear as clear zones against the lawn of living bacteria, called plaques. Lysogenic infection is initiated by temperate viruses and does not immediately result in the lysis and cell death of the host cell. The virus incorporates its genetic material into the host cell's genome. The host cell reproduces and its daughter cells contain the new copies of viral genetic material. With lysogenic infection, there will be a confluent lawn of bacteria with no plaques present.
What were the results of Fred Griffith's experiment?
Mice injected with harmless living R cells and heat-killed S cells died
You need to make 250 ml of a 20X stock solution of NaCl to dilute later to a final concentration of 50 mM. The M.W. of NaCl is 58.44 g. How you accomplish this?
NaCl = 14.61 g Pure Water = 250 mL 250 ml = 0.25 L 50 mM = 0.05 mol/L NaCl M.W. = 58.44 g (0.25 L)(58.44 g)(0.05 mol/L)(20) = 14.61 g
Describe the term "obligate intracellular parasite".
Obligate intracellular parasites are incapable of reproducing outside their host cell, meaning that the parasite's reproduction is entirely reliant on intracellular resources.
What are RFLPs and why can they be used for DNA fingerprinting?
RFLPs are restriction fragment length polymorphism, which are differences in the restriction sites on homologous chromosomes that result in different restriction fragment patterns. Loss of a restriction site results in the appearance of a larger fragment and the disappearance of two smaller fragments. Formation of a restriction site results in the disappearance of a larger fragment and the appearance of two smaller fragments. RFLPs can be used in DNA fingerprinting to convict felons, exonerate the innocent, and establish familial relations.
What does the RecA gene code for?
RecA gene codes for a protein, RecA, that regulates both bacterial recombination of chromosomal DNA and the expression of various mechanisms, which repair damage to DNA caused by UV radiation and other environmental agents
Why are restriction enzymes also called restriction endonucleases?
Restriction enzymes are also called restriction endonucleases because they cleave DNA within the molecule.
What does a lower agarose % in the gel do?
Separates larger fragments of DNA more easily
What bacteria is used in Fred Griffith's 1920s experiment?
Streptococcus pneumoniae
Why is E. coli strain B used in experiment 21?
T4 phage is specific to E. coli strain B
Assume you count 250 colonies on the +pGLO LB/amp/ara plate and a confluent lawn of bacteria on the +pGLO LB/amp plate. Did you expect this results? How could you prove your theory?
The + sign indicates that the plasmid was inserted into the bacterial cell. The transformed bacterial cells will be able to survive in the presence of ampicillin. However, the given results are not expected as there should be no difference in growth pattern provided the transformed culture plated is the same. Arabinose is a sugar which activates the gene for GFP production. On LB/amp plate, there will be growth with no fluorescence due to the absence of arabinose. On LB/amp/ara plate, there will be growth with fluorescence. They differ only in fluorescence, but both plates should show the same amount of growth.
Plaque
The area on the lawn of bacteria where the remnants of the dead, lysed bacterial cells will appear as a clear zone against the lawn of living bacteria
What kind of growth should be present when the bacterial host is added to an agar medium in experiment 21?
The concentration of the bacterial host should be sufficient enough to form a "lawn" of bacteria on the agar surface
What would you see if you repeated your lambda phage fingerprinting experiment with human DNA?
The human genome is extremely long with around 3 billion base pairs, so when cut with a restriction enzyme and the fragments are separated via gel electrophoresis, there will be no clear restriction pattern visible. Instead, only a smear of DNA representing fragments of just about every possible size will be visible.
Transformation
The transfer of genetic material from one bacterium to another, whereby one bacterial cell takes up the naked DNA from the environment
What is the equation used to calculate titer?
Titer = number of plaques/(quantity of virus sample (mL)) X (virus dilution)
You repeat the transformation experiment using E. coli auxotrophs instead of B. subtilis auxotrophs. What would be the outcome?
Unlike B. subtilis, E. coli is a naturally incompetent bacterium and would require an artificial transformation procedure. If the previous transformation experiment were to be conducted on E. coli, I would expect to see no growth because E. coli is unable to transform via natural transformation. In order for E. coli to grow on the GMS plate, the cell membrane would need to be made more permeable so it can incorporate itself with the donor DNA.
Agarose gel electrophoresis
Used to separate DNA fragments based on size
You repeat the viral plaque assay adding 10 microliters of viral solution to 990 microliters of LB. You perform this serial dilution five times. You add 10 microliters from each dilution to the corresponding host bacteria molten top agar mixture which is then spread onto an LB agar plate. The next day you count 39 plaques on the fourth plate. a) What is the concentration of the viral stock solution? b) What would you expect to see on the third and the fifth plate?
a) dilution factor = 1:100 10 microliters = 0.01 ml titer (pfu/ml) = # of plaques/virus sample (ml) x (dilution factor) = 39/(0.01)(10^-8) = 3.9 x 10^11 pfu/ml b) The third plate may have ~3900 plaques since it is 100 times more concentration than the fourth dilution. The fifth plate is very diluted, so I would expect to see little to no plaques.
You are given a T4 bacteriophage stock and asked to calculate the concentration. You perform a serial dilution by adding 10 microliters of the stock to 990 microliters of LB (dilution 1) and repeat this two more times. From your last dilution you add 100 microliters into a molten top agar tube containing 100 microliters of overnight E. coli strain B cells, vortex, and pour onto an LB agar plate. You incubate the plate at 37 degrees Celsius for 24 hours. The next day, you count 42 plaques. a) What is the concentration of the viral stock solution? b) The concentration of virus particles is referred to as the ___________.
a) titer (pfu/ml) = # of plaques/viral sample (ml) X dilution factor = 42 pfu/(0.1 ml)(10^-6) = 4.2 x 10^8 pfu/ml b) titer
What is the plasmid that is used in experiment 20?
pGLO plasmid
You serial dilute a virus sample using 100 microliters of virus into 900 microliters of LB (= a dilution factor of 1:10). 100 microliters of each dilution is added to some bacterial host cells and distributed onto agar plates. There are 80 plaques visible on the fifth plate following incubation. Calculate the titer of the fifth plate.
titer (pfu/ml) = # of plaques/qty. of virus sample (ml) x virus dilution 80 pfu/0.1 ml x 10^-5 = F