Module 5 - Bacterial Genetic Analysis Flashcards
What types of bacteria has scientists focused on for science?
Pathogenic bacteria, or ones with practical importance
What are some examples of bacteria that would be commonly studied?
E. coli found in the gut, salmonella species
What are some applications of bacterial genetics?
Industrial applications, Streptomycin antibiotics, E. coli protein production, and Pseudomonas decontamination
How is Streptomycin used in bacterial genetics applications?
It can create antibiotics
How is E. coli used in bacterial genetics applications?
It can produce useful proteins
How is Pseudomonas used in bacterial genetics applications?
It can be used for decontamination by breaking down toluene
True or false: microbial genetics is important in the study of microbiology
True: it is important to understand the natural genetic tools for genetic research
What is growth?
Increasing the number of cells (not size)
In which organisms is it easier to detect mutations: bacteria or eukaryotes?
Bacteria
How come it is easier to detect mutations in bacteria?
They have one copy of a gene, so a mutation can more easily be seen in the phenotype
How come it is harder to detect mutations in eukaryotes?
They have two copies of a gene, so a mutation can be masked by the other, functioning gene
In the first half of the 20th century, what was the belief about microbial genetics?
Microbes were too small to have genetic exchange
Before bacteria, what organisms were shown to have genetic exchange?
Corn, peas, and paramecium protozoa
What did the study of genetic exchange in eukaryotic organisms show?
Inheritance followed a sexual event
What do different strains of bacteria mean?
Genetically different cells (in the same species) with different phenotypes
What organism did Lederberg use for his experiments?
E. coli
What type of E. coli did Lederberg use in his experiments?
Different mutant nutrient strains, which had different metabolic requirements
What is an auxotroph?
A mutant strain having nutritional requirements additional to the wild type organism
What type of media would an auxotroph need?
Basic media, plus extra vitamins or amino acids
What is a prototroph?
A parent strain (to an auxotroph) that is able to grow just on basic media
What type of media would a prototroph need?
Basic media
What is found in basic media?
A source of carbon, nitrogen, and phosphorus (no additional vitamins or amino acids)
How come auxotrophs cannot grow in basic media?
They have a mutation that prevents them from making a specific vitamin or amino acid
In Lederberg’s experiments, which organisms could grow in medium with both methionine and proline?
All of the strains
In Lederberg’s experiments, which organisms could grow in medium without methionine or proline?
Only the prototrophic strain (met+, pro+)
In Lederberg’s experiments, which organisms could grow in medium with just methionine?
The prototrophic strain (met+, pro+), and the methionine auxotroph (met-, pro+)
In Lederberg’s experiments, which organisms could grow in medium with just proline?
The protorophic strain (met+, pro+), and the proline auxotroph (met+, pro-)
What is the makeup of a prototroph in Lederberg’s experiments?
Met+, pro+
What is the makeup of a methionine auxotroph in Lederberg’s experiments?
Met-, pro+
What is the makeup of a proline auxotroph in Lederberg’s experiments?
Met+, pro-
How can auxotrophs be used to see if bacteria can exchange genetic information?
If phylogeny of two auxotrophs can grow in general media, then there must be genetic exchange
True or false: if the phylogeny of two auxotrophs can grow in general media, there must be genetic exchange
False: reversion could have also occured
What is reversion?
A spontaneous mutation that corrected the metabolic deficiency
What are the chances of reversion for a single mutant?
10^-6 to 10^-7 for 10^8 cells
If 10^8 single mutants are plated, how many will rever?
10-100
What can be used to overcome the probability of reversion?
Double and triple mutant strains
What are the chances of reversion for a double mutant?
10^-12 to 10^-14 for 10^8 cells
What are the chances of reversion for a triple mutant?
10^-18 to 10^-21 for 10^8 cells
What happened when Lederberg crossed two triple mutant auxotrophs?
There were still some phylogeny that could grow in simple media
What was the conclusion of Lederberg’s experiment with triple mutant auxotrophs?
Genetic information must have been exchanged (reversion was very unlikely)
What did Lederberg show with cell extract?
Genes were not just taken up from dead cells
What was the conclusion of Lederberg’s follow-up with cell extract?
Live cells must have transferred the genetic information
Which is bigger: eukaryotic DNA or bacterial DNA?
Eukaryotic DNA
True or false: most of the eukaryotic DNA is transcribed
False: most of it does not encode for proteins
True or false: most of the bacterial DNA is transcribed
True: most of it is transcribed
What is the structure of DNA in most bacterial cells?
One circular chromosome
How else can DNA be arranged in a bacteria (abnormally)?
As linear chromosomes, or having more than one chromosome
Which bacterial species have linear chromosomes?
Streptomyces and Borrelia
What is a plasmid?
A circular DNA molecule that can replicate independently of chromosomal DNA
What are genes on a plasmid usually used for?
Survival of bacteria
What are some examples of genes found on plasmids?
Antibiotic resistance, toxic chemical degradation, or symbiosis
What trait is found on the R1 plasmid?
Antibiotic resistance
Where is R1 plasmid found?
Salmonella Paratyphi
What trait is found on the pSym plasmid?
Nitrogen-fixing nodule formation on legume plant roots
Where is pSym plasmid found?
Rhizobium
What trait is found on the pTi plasmid?
Tumor formation on plants
Where is pTi plasmid found?
Agrobacterium
What trait is found on the pTol plasmid?
Toluene degradation
Where is pTol plasmid found?
Pseudomonas putida
What trait is found on the pR773 plasmid?
Arsenic resistance
Where is pR773 plasmid found?
Escherichia coli
What trait is found on the pWR100 plasmid?
Entry into host cells
Where is pWR100 plasmid found?
Shingella flexneri
What plasmid does Salmonella Paratyphi have?
R1
What plasmid does Rhizobium have?
pSym
What plasmid does Agrobacterium have?
pTi
What plasmid does Pseudomonas putida have?
pTol
What plasmid does Escherichia coli have?
pR773
What plasmid does Shingella flexneri have?
pWR100
Which plasmid gives antibiotic resistance?
R1
Which plasmid gives nitrogen-fixing nodule formation on legume plant roots?
pSym
Which plasmid gives tumor formation on plants?
pTi
Which plasmid gives toluene degradation?
pTol
Which plasmid gives arsenic resistance?
pR773
Which plasmid gives entry into host cells?
pWR100
What is a genome?
All the genetic material in a cell, including plasmid DNA
What is a replicon?
DNA, region of DNA, or bacterial plasmid/chromosome that can replicate from a single origin of replication
What range can copy number take?
One to several hundred copies per cell
What controls copy number?
The initiation of DNA replication
What plays an important role in the evolution of bacterial chromosomes?
Bacteriophages and plasmid DNA
How come bacteriophages and plasmid DNA play an important role in the evolution of bacterial chromosomes?
They provide mechanisms of moving segments of the bacterial genome from one bacterial cell to another
What is an example of phage DNA in bacteria?
Clostridium botulinum toxin
What are incompatible plasmids?
Plasmids that cannot exist stably within a population
How can two plasmids become incompatible?
If they have a similar origin of replication
What happens if two plasmids in a cell have a similar origin of replication?
Replication will initiate randomly, so one of the plasmids will lose the count
How will replication control treat two incompatible plasmids?
As a single plasmid
What does random initiation of incompatible plasmids lead to?
Unequal numbers of the two plasmids
True or false: incompatible plasmids is a competitive mechanism to prevent replication of the other plasmid
False: it is a random selection that prevents replication of the other plasmid
What is a mutant?
A cell or strain possessing a mutation compared to a wild type strain
What is a mutation?
A change in the DNA sequence
What is an allele?
A different form of a gene (other)
What causes alleles?
Mutations
What are the outcomes of a mutation?
Loss-of-function, or gain-of-function
What is a genotype?
The collection of alleles of a given set of genes
How is a gene name written?
A 3 letter abbreviation in italics, followed by a capital letter
What are some examples of proper gene names?
lacZ and lacY (should be in italics)
What is the purpose of the capital letter in a gene name?
To separate genes that are in a common pathway (eg: lacZ and lacY)
How is a protein name written?
Same 3 letter abbreviation as the gene name, with the first letter characterized and no italics
What are some examples of proper protein names?
LacZ and LacY (no italics)
How can mutants be generated?
UV light or other chemicals
How are mutants identified?
Through changes in phenotype of growth patterns
What are some examples of phenotypes that can be used to identify mutants?
- Capsules –> mucoid / smooth colonies
2. Flagella / red pigmentation
What bacterial mutants can be identified by red pigmentation and flagella?
Serratia marcescens
How can potentially rare mutants be detected?
Through phenotypic selection or phenotypic screening
If 10^9 CFU are plated on media with proper growing requirements, how many cells are expected to grow?
10^9
What happens if 10^9 CFU are plated on media with streptomycin?
A majority of the cells will not grow, but a few mutants resistant to streptomycin will grow
What is commonly used for phenotypic selection?
A drug/antibiotic
What are the steps of phenotypic selection?
Plate the bacteria on a plate with a drug or antibiotic, and see which ones grow
What happens if mutants look different than wild type colonies?
They can be detected directly on a single plate
What happens if mutants look like the wild type colonies?
2 different conditions are needed to distinguish between the two
For phenotypic selection, what are the two conditions used?
All nutritional support, and lacking a particular nutrient
For phenotypic selection, if condition 1 has all the nutritional support, what is the result?
All cells (wild type and mutant) will grow
For phenotypic selection, if condition 2 lacks a particular nutrient, what is the result?
Only wild type cells will grow (the mutant cells will not grow)
For phenotypic selection, after having two conditions, which cells are the mutants?
Those that grow on the full support, but did not grow on the media lacking a nutrient
What are the steps of phenotypic screening?
Plate the bacteria in two different conditions, and compare which ones didn’t grow in the condition lacking a particular nutrient
Which is more efficient: screening or selection?
Screening
How come screening is usually les efficient than selection?
Large amounts of colonies need to be screened to find a rare phenotype
How can duplicate plates be created?
Through replica plating
What process is replica plating useful for?
Phenotypic screening (when phenotypic selection can not work)
What is replica plating?
The process of duplicating the colonies onto multiple plates
What is replica plating similar to?
Making a photocopy from a master copy
What are the steps of replica plating?
Colonies are lifted from a master plate using a sterile velvet cloth, and then replicas are made on new plates
True or false: all mutations are bad
False: while most can disrupt gene function, they can also be beneficial
What provides the raw material for environmental pressures to act?
Changes in genetic material (mutations)
What happens if a mutation in a clone is disadvantageous?
The population of mutants will be low
What happens if a mutation in a clone is advantageous?
It will compete with other cells, and the number of cells with that mutation will increase
What did Richard Lenski do?
Proved that mutations can be advantageous experimentally
What was Lenski’s experimental setup?
12 parallel cultures of E. coli were grown in a media, and frozen after every 75 days
What was present in the media of Lenski’s experiments?
Glucose as a carbon source
How were Lenski’s E. coli subcultured?
Once per day, into fresh glucose media
How long did the Lenski experiment last?
10,000 generations (1500 days)
What did Lenski do after the 10,000 generations?
The new cells were compared to the frozen ancestors
How come the ancestors were frozen in Lenski’s experiments?
To prevent them from evolving
What was the conclusion of Lenski’s experiments?
The ability for E. coli to grow in culture was enhanced over time
How did Lenski compare the new cells to the ancestral cells
He mixed them in equal ratio, and then plated them to see the proportion of each
How were the two strains in Lenski’s experiments differentiated?
By color (through a neutral marker)
How was the relative fitness calculated?
The ratio of red (new) to white (ancestral) colonies
What was the result of Lenski’s relative fitness calculations?
Both cell size and survivability increased in evolved cultures
In Lenski’s experiments, how were fitness gains formed?
From mutations in genes involved in the cellular stress response
What are fitness gains specific to?
Given environmental conditions
What did Ester Lederberg do?
Studied whether mutations can arise in the absence of selective conditions
What were the steps of Lederberg’s experiments on selection?
- E. coli were grown on a master plate without any drug
- Replica plates were made for plates with and without streptomycin
- Mutants from the antibiotic free plate were plated on streptomycin
What was the purpose of the first two plates in the Lederberg experiment on selection?
To identify colonies that were streptomycin resistant
What was the purpose of the third plate in the Lederberg experiment on selection?
To show that colonies were streptomycin resistant, even though they have not been subjected to streptomycin
What was the conclusion of Lederberg’s experiments on selection?
Mutations can occur in the absence of selective pressure
How did Lederberg’s results support her conclusion about selection?
Cells that were never exposed to streptomycin still developed resistance
What role do restriction enzymes play?
Molecular scissors
How do restriction enzymes work?
They recognize a specific DNA sequence, and make a double stranded cut
What is a restriction site?
The DNA sequence recognized and cut by the restriction enzyme
How many base pairs is a restriction site?
4, 6, or 8 bp
What is another name for sticky ends?
Cohesive ends
What is the result after restriction enzymes cut DNA?
Either sticky ends or blunt ends
What do sticky ends look like?
Overhangs
What do blunt ends look like?
Straight cuts
What does the name of the restriction enzyme indicate?
The source (which bacterium it came from)
What does the first letter of a restriction enzyme indicate?
The genus of the source
What do the second and third letters of a restriction enzyme indicate?
The species of the source
What does the fourth letter of a restriction enzyme indicate?
The strain of the source
What is the structure of restriction sites?
Palindromes
What is a palindrome?
A DNA sequence that is the same when read from both strands of DNA
How can DNA cut by restriction enzymes be joined together?
Through DNA ligase
What does DNA ligase do?
Reforms the phosphodiester bonds between adjacent 5’ phosphate groups and 3’ hydroxyl groups
What DNA can bind to blunt ends?
Any other DNA segment with blunt ends
Where does EcoRI come from?
Escherichia coli
Where does BamHI come from?
Bacillus amylolique faciens
Where does HindIII come from?
Haemophilus influenzae
Where does SmaI come from?
Serratia marcescens
Which restriction enzyme produces blunt ends?
SmaI
What is the restriction site for EcoRI?
5’ GAATTC 3’
3’ CTTAAG 5’
What is the restriction site for BamHI?
5’ GGATCC 3’
3’ CCTAGG 5’
What is the restriction site for HindIII?
5’ AAGCTT 3’
3’ TTCGAA 5’
What is the restriction site for SmaI?
5’ CCCGGG 3’
3’ GGGCCC 5’
How is the restriction site for EcoRI cut?
5’ G \ AATCC 3’
3’ CTTAA \ G 5’
How is the restriction site for BamHI cut?
5’ G \ GATCC 3’
3’ CCTAG \ G 5’
How is the restriction site for HindIII cut?
5’ A \ AGCTT 3’
3’ TTCGA \ A 5’
How is the restriction site for SmaI cut?
5’ CCC \ GGG 3’
3’ GGG \ CCC
What are the steps of generating recombinant DNA from restriction enzymes?
- Digest the DNA with the restriction enzyme
- Mix digested DNA and incubate
- Treat annealed DNA fragments with DNA liagse
What can restriction enzymes be used to create?
Recombinant DNA
How do DNA molecules cut with EcoRI anneal?
Based on complementary base pairing on the sticky ends generated from EcoRI
What is molecular (DNA) cloning?
The process of replicating recombinant DNA molecules
What is used for DNA cloning?
Cloning vectors
What is a cloning vector?
A DNA molecule that can be genetically manipulated or replicated within cells
What are some examples of cloning vectors?
Plasmids, cosmids, and phages
What does a vector do?
Inserts recombinant DNA molecule into a recipient host bacterial cell
When were plasmid cloning vectors first used?
1970
Who performed the first plasmid cloning experiments?
Stanley Cohen
What plasmids were used in the first plasmid cloning experiments?
pSC101 and pSC102
What does pSC101 contain?
A tetracycline resistance gene
What does pSC102 contain?
A kanamycin resistance gene
What does Cohen do with pSC101 and pSC102?
He digested them with EcoRI, and combined the fragments to create pSC105
What is pSC105?
A plasmid created from pSC101 and pSC102
What does pSC105 contain?
A tetracycline resistance gene, and a kanamycin resistance gene
What does blue-white screening allow for?
Visual differentiation of colonies that carry the insert or do not
What is the advantage of blue-white screening?
Screening is done all on one medium
What plasmid is commonly used for blue-white screening?
pUC18
What is pUC18 used for?
Blue-white screening
What does pUC18 contain?
An ori, an antibiotic resistance gene, and a lacZ gene
What does the lacZ gene of pUC18 code for?
The amino terminal fragment of the enzyme beta-galactosidase
What part of beta-galatosidase does E. coli produce (without the plasmid)?
The carboxyl terminal fragment
What happens if pUC18 is inserted into E. coli (unchanged)?
Functional beta-galactosidase is formed, which can break down X-gal
What does beta-galactosidase do?
Breaks down X-gal, which gives colonies a blue color
What color are cells with unedited pUC18?
Blue
What happens when a gene of interest is inserted into pUC18?
It disrupts the lacZ gene, so beta-galactosidase cannot be formed
What happens if pUC18 with a gene of interest is inserted into E. coli?
Beta-galactosidase cannot be formed, so they cannot break down X-gal
What color are cells with pUC18 with the gene of interest?
White
What does white cells mean in blue-white screening?
The bacteria took up the plasmid with the gene of interest
What does blue cells mean in blue-white screening?
The bacteria took up the plasmid, but not with the gene of interest
Where is a gene of interest inserted in pUC18?
In the middle of the lacZ gene
What traits (5) are desirable on plasmids for easier gene cloning?
Origin of replication, selectable marker gene, multiple cloning site, small size, and high copy number
True or false: any ori will work on a plasmid
False: the ori needs to work in the specific host cell
What types of genes are commonly found as selectable markers?
Antibiotic resistance
Why would researchers want a plasmid with small size?
It maximizes transfer
Why would researchers want a plasmid with high copy number?
Many DNA fragments can be made in a small number of cells
Why would researchers want a plasmid with a multiple cloning site?
It helps in cloning and screening of cells containing clone DNA
What is the most common vector used?
Plasmids
What is the reasoning behind phage vectors?
They take advantage of the ability of viruses to infect and deliver genomes into cells
How is a phage vector developed?
The DNA fragments that need to be cloned and phage DNA are mixed and ligated together
How big is the entire lambda phage genome?
~50 kb
What is done after the phage vector is developed?
It is added to E. coli on solid media to form plaques
What happens if a phage vector is integrated?
The phylogeny phages are not produced, so clone fragments will not be amplified
How is the problem of phage vectors being incorporated overcome?
By removing the genes for integration and excision (not essential for viral replication)
True or false: the integration and excision genes in phage vectors are necessary for viral replication
False: they can be removed from the genome
How big are the integration and excision genes?
~20 kb
Where is the DNA inserted in the phage vector?
Where the integration and excision genes used to be in the phage genome
What is a cosmid?
A hybrid plasmid that contains phage cos sequences
What is a cosmid a combination of?
A cos site and a plasmid
What does a cos site do?
It is required for packing into phage particles
Where are cosmid DNA sequences originally from?
The lambda phage
Once the DNA is in the host cell, how is the cosmid maintained?
As a plasmid
How big of an insert can most plasmids handle?
Up to 15 kb
How big of an insert can phages handle?
Up to 24 kb
How big of an insert can cosmids handle?
Up to 45 kb
What is the composition of a cosmid?
A cos site, a multiple cloning site, and an antibiotic selection marker (plus the gene of interest)
How come a cosmid has a lot of room for insert DNA?
Nearly all of the phage DNA is omitted
What is horizontal gene transfer?
The movement of DNA between microbes
True or false: horizontal gene transfer is a major contributor to evolution
True: it can facilitate the movement of DNA
When DNA enters a cell, what are its two options?
Either replicate on its own (plasmid), or join the host cell’s DNA
What is recombination?
The incorporation of foreign DNA into host cell’s chromosomes
What does recombination involve?
The breaking and joining of two DNA strands
What are the two forms of recombination?
Homologous and non-homologous recombination
What is homologous recombination?
When 2 identical (or nearly identical) fragments of DNA line up and exchange pieces
What is another name for homologous recombination?
Crossover
In what organism is homologous recombination best understood?
E. coli
Which enzymes are present in homologous recombination?
The RecBCD complex and the protein RecA
What structure is produced during homologous recombination?
The Holliday junction
What is the structure of the Holliday junction?
An X shape, where the 2 DNA molecules are linked together
What happens when the Holliday junction is cleaved?
The DNA is separated such that recombined DNA molecules are formed
What does RecBCD do?
Unwinds and nicks the DNA fragment
What does RecA do?
Bind single stranded DNA to scan for homology. It then produces a nick, and joins the strands together
What is strand exchange?
When the two DNA strands are swapped by RecA in homologous recombination
What happens if there is a single crossover between two circular molecules?
One circular molecule is produced (integration)
What happens if there is a double crossover between two circular molecules?
There is reciprocal exchange of the region between the 2 crossover events (DNA swap)
What happens if there is a double crossover between a circular molecule and a linear molecule?
Reciprocal exchange of the DNA from first molecule into the second molecule (integration) between the 2 crossover events
Where does non-homologous recombination take place?
In all forms of life
Where is non-homologous recombination most common?
Viruses and transposable elements
What is non-homologous recombination?
Recombination of DNA pieces with little or no sequence similarity
How do viruses use non-homologous recombination?
They can integrate their genome into the host chromosome
Where does integration of the viral genome into the host chromosome occur?
At specific, but non-homologous, sites
What enzyme allows for the viral genome to be integrated into the host chromosome?
Integrase
What does integrase do?
Allows the viral genome to be integrated into the host chromosome
What is transformation?
The introduction of extracellular DNA (plasmid/DNA fragment) directly into an organism
True or false: transformation requires cell/cell contact
False: transformation does not require cell/cell contact for DNA uptake
What bacteria are naturally competent to take up DNA from their surroundings?
Bacillus, Streptococcus, Haemophilus, and Neisseria
How come Bacillus and Streptococcus are naturally competent to take up DNA from their surroundings?
They have specialized machinery for the uptake of DNA
Which bacteria are not naturally competent to take up DNA from their surroundings?
E. coli
How can cells like E. coli be made competent for transformation?
Through adding calcium ions or through electroporation
How does a solution of cations like calcium aid in transformation?
It makes the membrane more permeable to large molecules, such as DNA
What is electroporation?
Applying an electric current that generates holes in the cellular membrane for DNA to enter
What is the advantage of electroporation?
It can be applied to a broad range of bacteria
What is the first step of transformation for naturally competent cells?
DNA binds to a DNA binding receptor on the cell surface
What happens once DNA binds to a DNA binding receptor?
One strand is degraded, and the other strand transported through a specialized channel pore
What happens once DNA enters a cell through the DNA binding receptor?
It can either be used as nutrients, or integrated into the host chromosome through recombination
What enzyme pulls DNA into the cell?
DNA translocase
What does DNA translocase do?
Pulls DNA into the cell
What happens to the single stranded DNA once it enters the cell?
It binds to single stranded DNA binding proteins and RecA
What is conjugation?
Transfer of DNA from cell/cell via direct contact/sex pilus formation
True or false: conjugation can only occur within one domain
False: conjugation can occur between domains
True of false: conjugation can lead to human pathogens
True: conjugation can result in the conversion of non-pathogenic organisms into human pathogens
What does conjugation in E. coli require?
The fertility factor F
What is the fertility factor?
A circular double stranded DNA plasmid called the F plasmid
What are F+ strains?
Stains that have the F plasmid
What can F+ strains do?
Can donate DNA to F- strains
What happens when an F+ strain donates DNA to an F- strain?
The F- strain becomes an F+ strain
What does the F plasmid contain?
All genes needed for conjugation and plasmid maitenance
What does tra region stand for?
Transfer region
What is found in the tra regions?
Genes for proteins needed for production of conjugational structures that link cells together and transport DNA
How can the F plasmid be copied?
Using the rolling circle method, and then sent across the bridge into the recipient cell
What mediates conjugation?
A special sex pili
What encodes the sex pili?
Some of the tra genes
What happens after a donor and recipient contact (in conjugation)?
A mating bridge forms
What is a mating bridge?
A complex of tra encoded proteins that facilitates transfer of DNA in conjugation
What happens in the donor cell once a mating bridge is formed?
An endonuclease makes a single stranded nick at the origin of transfer of the F plasmid
What happens once there is a nick in the F plasmid at the origin of transfer?
One strand of the F plasmid gets donated to the recipient cell through the mating bridge
What happens once each cell in conjugation has one strand of DNA?
DNA polymerase synthesizes the complementary strand
After the mating bridge, how many strands of DNA are found in each cell?
Each cell has one strand
What happens once DNA polymerase synthesizes the complementary strands of the plasmids in conjugation?
The ends of the plasmid release from the mating bridge, and attach to the plasmid to regain the circular form
Besides conjugation, what can the F plasmid do?
Integrate with the host chromosome
What type of recombination can occur between the F plasmid and the host chromosome?
Homologous DNA recombination
What type of crossover produces an HFR strain?
A single crossover event (incorporation)
What is an HFR strain?
A strain where the entire F plasmid incorporates into the chromosome
What does HFR stand for?
High frequency of recombination
What happens when the F plasmid in an HFR strain initiates transfer?
Attached chromosomal genes can also be transferred to the recipient cell
What is the significance of HFR strains?
They allow new opportunities to pass genetic information from one cell to another
How can researchers use HFR strains?
They can be used to map locations of genes in the host chromosome
How can HFR strains be used to map locations of genes on the host chromosome?
Multiple auxotrophic mutants can be monitored for regained function over time of conjugation
True or false: HFR strains convert F- strains into F+ cells
False: the recipient cell remains an F- cell
How come HFR strains do not convert F- strains into F+ strains?
Only a part of the host chromosome (F plasmid) is transferred into recipient cells
What happens when a mating bridge is formed in HFR strains?
The origin of transfer is nicked, and only part of the F plasmid is transferred
For the entire F plasmid to be transferred in an HFR strain, what would need to happen?
The entire chromosome would need to move into the recipient cell
How come it is rare for an HFR strain to transfer the entire F plasmid?
Mating pairs often separate before the entire chromosome can be transferred
Which genes in an HFR strain are donated at the highest rate?
Those near the site of F insertion
Which genes in an HFR strain are donated at the lowest rate?
Those further from the site of F insertion
How is an F’ plasmid formed?
Recombination occurs between either side of F insertion, resulting in looping out of a segment of the chromosome
What is an F’ plasmid?
An F plasmid that also contains large amounts of chromosomal DNA
What is an F’ strain?
A strain that contains an F’ plasmid
What genes (generally) do F’ strains donate, and how frequently?
They transfer only a limited number of genes, but do so at extremely high frequency
What happens when F’ strains mate with F- strains?
They transfer the modified F’ plasmid into the recipient cell
What happens to the transferred chromosomal DNA in F’ strains after conjugation?
It remains part of the F plasmid in the recipient cell
What happens to the host chromosome after an F’ plasmid is created?
There is a deletion of the the genes (they are not found on the F’ plasmid)
What is transposition?
Movement of DNA via mobile genetic elements (transposable elements)
What do transposable elements do?
Mediate gene arrangements by moving within and between genomes
Where were transposable elements first discovered?
In corn by Barbara McClintock
What did Barbara McClintock do?
First discovered transposable elements in corn
True or false: transposable elements are limited to a few species
False: they are found in virtually all genomes
What are simple transposable elements?
Sequences that only encode for proteins necessary for transposition
What is another name for simple transposable elements?
Insertion sequences, or IS elements
What does IS elements stand for?
Insertion sequences
What are complex transposons?
Sequences that encode for proteins necessary for transposition as well as other genes (such as antibiotic resistance)
What does Tn stand for?
Transposon
Where do transposable elements insert themselves?
Randomly into the genome
How do many researchers use transposable elemtns?
By using them to generate mutations to disrupt the function
How to transposable elements disrupt gene function?
By inserting themselves within a gene, thus disrupting the sequence and consequently the function
What is needed for transposon mutagenesis?
A suicide vector plasmid
What is a suicide vector?
A transposon carrying plasmid that is unable to replicate inside the cell
How are transposon insertion mutants isolated?
By using an antibiotic resistance transposon on a suicide vector
What is selected for in transposon insertion mutants?
The antibiotic resistance transposon to move from the suicide plasmid (lost from the cell) to the host chromosome
What is transduction?
The transfer of bacterial DNA from one cell to another by a bacteriophage
How do phages work?
They infect bacteria, and use cellular machinery to copy themselves
What can happen during viral packaging in a host cell?
Segments of genomic DNA from the infected bacterial cell can become packaged into the virus capsid
What are transducing particles?
Phages that carry bacterial DNA
True or false: transducing particles are effective in infection
False: they are usually not effective in infection
How come transducing particles are not effective in infection?
They usually lack phage genes needed to form infectious viral particles
True or false: transducing particles cannot transmit their DNA
False: they can attach to recipient cells and inject the DNA
What happens to DNA injected from a transducing particle?
It can undergo homologous recombination to insert into the recipient genome
What is a transductant?
Bacteria that receive DNA through transduction
What is the host range of phages?
Very narrow
In what circumstance is transduction particularly efficient?
Transferring DNA between strains in the same species
What is needed for incorporation in transduction?
Homologous recombination
True or false: transducing particles can replicate
False: they cannot replicate
How come transducing particles cannot replicate?
They lack the viral genome
Why are Bacillus commonly studied?
They create endospores
When did microbial genetics first start?
The mid 20th century
Before microbes, what were the model organisms for genetics?
Fruit flies, corn, and peas
What are some advantages of bacteria as a genetic organism of study?
- Each cell is a complete organism (growth = more cells)
- Simple cellular differentiation
- Simpler chromosome organization
If 10^8 double mutants are plated, how many will revert?
0.0001-0.00001 colonies per plate
If 10^8 triple mutants are plated, how many will revert?
Virtually no colonies
What does it mean if a genome is “genetically compact”?
Most of the DNA encodes functional proteins
What genes are found in the host chromosome of bacteria?
Genes for basic metabolic processes of the organism
Which species of bacteria have more than one chromosome?
Agrobacterium, Vibrio cholerae, and Burkholderia
What chromosomes does Agrobacterium have?
One linear, one circular
What chromosomes does Vibrio cholerae have?
Two circular
What chromosomes does Burkholderia have?
Three circular
What is copy number?
The controlled number of plasmid molecules in a cell
What controls the copy number?
Initiation of DNA replication
True or false: DNA polymerase acts at a constant rate
True: it is not responsible for copy number since it replicates at a constant rate
How come DNA polymerase does not affect copy number?
It replicates at a constant rate
For two or more plasmids to exist inside a cell, what needs to happen?
They need to be in different incompatibility groups
What is a wild-type strain?
A strain that possesses the typical or representative characteristics of the species
How can a wild-type strain be first isolated?
Either in nature, or the common lab strain that mutants are derived from
What is the wild-type strain for E. coli?
E. coli K12
Where and when was the wild-type strain of E. coli isoalted?
From human feces in 1922
Where was the wild-type strain of Sinorhizobium meliloti isolated from?
An antibiotic-resistant derivative of an isolate from an alfalfa root nodule
What happens if a new strain is identified?
It is archived and cataloged in freezers in central strain locations
What is the common name for HisC?
Histidinol phosphate aminotransferase
What does HisC do?
It is involved in the biosynthesis of histidine
What types of phenotypes are selection used for?
Ones where the mutant can grow and the wild type can not
What types of phenotypes are screening used for?
Ones where the wild type can grow and the mutant can not
What is an alternative to the velvet cloth method for replica plating?
Using a toothpick and a grid to transfer colonies
What are the advantages of using a toothpick and grid over a sterile velvet cloth?
It has greater precision and reproducibility
What are bacterial colonies composed of?
Millions of genetically identical organisms (clones)
When did Lenski first start his experiments?
February 15th, 1988
What does LTEE stand for?
Long Term Evolution Experiment
What was the purpose of Lederberg removing cells distant from the antibiotic resistant plate in the selection experiments?
It served as a control that not all the bacteria were resistant to streptomycin
What did Salvador Luria and Max Delbruck do?
Investigated whether mutations occurred randomly and spontaneously
Who continued Lederberg’s idea about mutations occuring randomly?
Salvador Luria and Max Delbruck
What was the rationale behind Luria’s and Delbruck experiment?
E. coli resistant to T1 phage would pass it on to their offspring. If they developed it earlier, more phylogeny would have the resistance
What experiment did Luria and Delbruck run?
The fluctuation test
What are the steps of the fluctuation test?
- Inoculate a large flask with E. coli, and plate onto plates with T1
- At the same time, inoculate independent cultures, and plate each onto plates with T1
What were the results of the large flask from the fluctuation test?
Similar numbers of resistant colonies were seen in the plates
What were the results of the independent cultures from the fluctuation test?
Each plate had a variable amount of resistant colonies
How come independent cultures led to variable amounts of resistant colonies in the fluctuation test?
Each colony developed resistance at a different point, and thus more or less bacteria would gain resistance
How come the large flask led to similar amounts of resistant colonies in the fluctuation test?
The mutants were evenly distributed in the large culture
What was the conclusion of the fluctuation test?
Mutations occur spontaneously and randomly in the absence of selective pressure
What were the sequence of advancements towards recombinant DNA?
- 1944 - DNA was shown to be the heritable material
- 1960s - DNA could be extracted from cells
- DNA ligase was extracted
- 1970 - restriction enzymes were discovered
- 1973 - first recombinant DNA was made
When was the first recombinant DNA molecule made?
1973
Who showed the DNA was the first heritable material?
Avery, MacLeod, and McCarthy
What does a Roman numeral refer to in the name of a restriction enzyme?
Its order of isolation from that strain
What was one of the first cloning vectors used?
pBR322
What is contained in pBR322?
Ampicillin resistance, tetracycline resistance, unique restriction sites, and a small size (4.36 kb)
How are linear and circular DNA molecules selected against in transformation?
DNA without an ori will be degraded and lost from the cell
If an alpha-amylase gene is inserted into pBR322, what steps are done to screen for it?
- Do a general transformation on ampicillin for ampicillin resistance
- Do a screening on tetracycline for tetracycline sensitivity
- Do a functional analysis on starch breakdown for amylase activity
What is a multiple cloning site?
Short segments on the vector that contain a cluster of different restriction enzyme sites that only appear once in the plasmid
What is the advantage of a multiple cloning site?
It makes it easier to clone the vector by only having one place to cleave
What does blue-white screening combine?
Antibiotic resistance screening with visual differentiation screening
What are most of the plasmids used in molecular biology derived from?
ColE1 from E. coli
What ori does ColE1 have?
OriV
What does oriV do?
It is an origin of replication only for E. coli and closely related species
What are shuttle vectors?
Vectors that can replicate in a more diverse range of hosts
How do shuttle vectors work?
They have multiple oris so they can replicate in multiple hosts
What is one use for shuttle vectors?
The in vivo assembly of synthesized overlapping oligonucleotide fragments in yeast
How do yeast function in synthesizing DNA fragments?
In yeast, DNA fragments can form one, continuous fragment, which can be ligated into the shuttle vector and inserted into E. coli
What is the advantage of using yeast to synthesize DNA fragments?
It can produce very large fragments of DNA with minimal in vitro steps
What is needed for DNA to be packages correctly in a phage vector?
A phage cos site at both ends of the linear fragment
What happens once the DNA fragment enters the capsid head?
A viral endonuclease cleaves the DNA at each cos site, leaving single strand overhangs that can recircularize the DNA once in the cell
What is vertical gene transfer?
The inheritance of a gene from a direct ancestor
What is recombination a spin-off of?
The DNA repair mechanism
What happens once the Holliday junction is formed?
Two nicks are made to produce two recombinant DNA molecules
What is the result of a single crossover between a circular chromosome and a linear DNA fragment?
A single linear molecule, which is not stable and thus degraded
What is lytic replication?
New copies of the virus are produced within the cell
What is lysogenic replication?
The phage genome becomes integrated into the host chromosome
What is site-specific recombination?
Non-homologous recombination at specific sites (usually seen in viral replication)
What is a knockout mutant?
A mutant where the function of a gene is disrupted
True or false: a suicide vector has no ori
False: it usually has an ori for the cloning cell, but not the host cell
What is a cointegrate?
The joining of a plasmid and the chromosome as one unit
What are the advantages and disadvantages of using a double crossover gene knockout?
They lead to more stable insertions, but they are harder to make
What does sacB do?
It leads to sensitivity to sucrose by producing levansucrase
What does levansucrase do?
Hydrolyzes sucrose
What happens if a sacB gene is active in a cell?
The cell will die in the presence of sucrose
How can sacB be used in a double crossover gene knockout?
It can be used to ensure proper crossover (kanamycin and sucrose resistant mutants have the gene of interest)
In a double crosover gene knockout, where would the sacB gene be found?
On the suicide vector
What is an example of transformation being used by researchers?
The studies by Fredrick Griffith in the 1920s
What did Fredrick Griffith do?
Studied how Streptococcus could become pathogenic through transformation
Who demonstrated that gene transfer in conjugation occurred in only a single direction?
William Hayes
What did William Hayes do?
Showed that gene transfer in conjugation occurred in only a single direction
What happens if there is a loss-of-function of the tra genes?
There is a loss of F plasmid transfer function
What did Bernard Davis do?
Used immersed filters that prevented cellular contact to study conjugation
Who studied if conjugation required cellular contact?
Bernard Davis
What does oriT stand for?
Origin of transfer
How does the F plasmid integrate into the host chromosome?
Through a single-crossover event
What is a transductant?
A cell that has incorporated DNA from another cell via conjugation
How does a transductant incorporate DNA?
Through homologous recombination
How can a gradient of transfer for HFR strains be tested?
Through purposeful interruption of mating during conjugation
Before DNA sequencing was available, how were genes mapped in E. coli?
Through a gradient of transfer of HFR strains
How long does it take for an entire chromosome to be transferred during conjugation
A constant rate of 100 minutes for the entire chromosome to be transferred
For the E. coli K12 genome, what is the rate of transfer?
46 kbp per min
How were F’ plasmids used in research?
Strains can be constructed that carry two different gene alleles or copies of a chromosomal region
How would an F’ plasmid be used to make two alleles?
One allele would be in the host chromosome, and the other would be on the F’ plasmid
What is a merodiploid?
A partial diploid that has two copies of some of its DNA
Why did triparental conjugation need to be derived?
Conjugation requires oriT and tra genes, but this would make the plasmid too large
How does triparental conjugation work?
The tra genes are provided by a helper plasmid, while the donor strain has the recombinant plasmid to be donated to the recipient cell through conjugation
What is the first step of triparental conjugation?
The helper strain transmits the helper plasmid into the donor strain
What is the second step of triparental conjugation?
The helper plasmid expresses the tra genes to allow the recombinant plasmid to move from the host to the recipient through conjugation
How can the recipient cell be isolated in triparental conjugation?
By using media where none of the three original strains can grow
What is needed for transposition to occur?
Transposase and terminal inverted repeat sequences
What does the transposase enzyme do?
Recognizes terminal inverted repeat sequences and performs single-stranded cleavage
What is non-replicative transposition referred to as?
Cut-and-paste
What is the result of non-replicative transposition?
Transposable elements re excised from one location and inserted into another
What is replicative transposition referred to as?
Copy-and-paste
What is the result of replicative transposition?
It requires a replication step to leave the transposable element at the original site
What do res sites do?
Site for resolvase to separate joined DNA molecules
What does resolvase do?
Separates joined DNA molecules in replicative transposition
What happens in non-replicative transposition?
There is repair of single-stranded gaps by DNA polymerase and DNA ligase
What happens in replicative transposition?
Replication of the transposon by DNA polymerase results in joining of two DNA molecules, which is separated by resolvase
What happens once transposase recognizes the sequences?
There is a joining of the cleaved strands to form an intermediate
What is the significance of terminal inverted repeat sequences?
Site that transposase recognizes
What sites does transposase recognize?
Terminal inverted repeat sequences
What is the composition of IS elements?
A transposase gene flanked by two terminal inverted repeat sequences
What are terminal inverted repeat sequences?
Short sequences of DNA that exist in an inverted orientation relative to each other
What type of transposition can IS elements undergo?
Non-replicative transposition
What is needed in the transposon to undergo replicative transposition?
A resolvase gene and a res site
Which genes have often been associated with transposons?
Antibiotic resistance genes
True or false: transposons have target site specificity
False: transposable elements can insert anywhere in the genome
What does homologous recombination of transposons lead do?
Deletions, inversions, and translocations
How do most researchers use transposons?
To generate mutations in a bacterial cell
How were transposons used in the 2008 study in Alabama?
The transposon was used to create mutations, and the places that the transposon inserted at the cell survived were deemed not necessary for survival
Which transposon was used in the 2008 study in Alabama?
Tn4001T
What organism was studied in the 208 study in Alabama?
Mycoplasma pulmonis
How are transposition mutants isolated?
Through a suicide vector and antibiotic screening
Who first discovered transduction?
Norton Zinder
What did Norton Zinder do?
First discovered transduction
What was the first organism used to discover transduction?
Salmonella typhimurium
What bacteriophage was used to discover transduction?
P22
What experiment did Zinder do?
He observed prototrophic recombinants resulting from mixing two auxotrophic strains, even when a filter was applied
What are the steps for using transduction in a lab?
- Culture bacteria with a small amount of lysate
- Collecting the resulting viral particles
- Use these particles for the recipient strains
How does DNA in transduction integrate into the host cell genome?
Through double crossover recombination
What is cotransduction frequency?
The frequency with which an unselectable marker gene is transduced along with a given selectable marker gene
What is cotransduction used for?
Mapping genes
What does cotransduction frequency depend on?
The distance between marker genes
How was transduction used to combat HIV?
CCL3 and CCL5 genes were transduced into Latobacillus gasseria bacteria
What bacteria was targeted for HIV treatment?
Latobacillus gasseria
Where is Latobacillus gasseria found?
In the vagina
What do CCL3 and CCL5 do?
Potentially combat against HIV
