Module 6 - Notes Flashcards
How would you engineer a bacteria to use glucose under low cellular density and switch to succinate or pyruvate at high density?
Any of the following:
Have a quorum sensing molecule that prevents transport or utilization of glucose at high density
Have a quorum sensing molecule that induces transport or utilization of succinate/pyruvate at high density
How do bacteria acquire genetic diversity?
Evolution & adaptation
New mutations (Vertical)
Gene Exchange (Horizontal)
Gene Acquisition (Horizontal)
Selfish genetic elements (horizontal)
What are mutations?
Change in the nucleotide sequence.
They are heritable (Passed down)
They can be wild-type - isolated from nature (parent = starting strain)
The mutant (Child)
carries change and has a different genotype
What are the results of mutations?
Neutral - No observable change
Beneficial - Gain function
Detrimental - Lost function
Does the change in genotype result in a change of phenotype?
Not always
the phenotype might stay the same or it may result in a change of behaviour/protein activity
What are spontaneous mutations?
happen for unknown reasons
For every million to 10 million base pair replications, there will be 1 error at 1 base pair resulting in a silent mutation.
What is an induced mutation?
Caused by stresses
- nutrition, oxygen, etc.
Caused by mutagens
- Naturally or in the laboratory
- Example, UV light
How do we isolate gain of function mutants?
Selection
Change is beneficial
Select by phenotype
ie. antibiotic resistance
If the mutation is selectable you can find the mutation fairly quickly
How do we isolate non-selectable mutations? Those that are neutral or detrimental?
Screening
- a slower and tedious process
Following mutagenesis, you need to isolate antibiotic-resistant bacteria. What type of procedure(s) will you use?
A. Selection
B. Screening
C. Selection & Screening
D. Screening then Selection
A. Selection
How do we determine mutagenic potential?
Ames test
The ability of chemical to induce revertant in an auxotroph
The higher the number of colonies, the higher the potential
After an Ames test, you have the following revertants on the plate:
2 for the control
100 on treatment #1
1000 on treatment #2
2 on treatment #3
Which treatment has the lowest mutagenic potential?
Treatment #3
What are the types of effects of single-point mutations?
Silent
Missense
Nonsense
What is the molecular basis of point mutation?
substitution of a single base pair
Affects protein function
What is a silent mutation?
Substitution of a 3rd base codon (wobble position)
No change in the amino acid
What is a Missense Mutation?
Substitution of 1st and 2nd base of codon
Amino acid change in protein
What is a Nonsense Mutation?
Amino Acid change to a stop codon
Incomplete/truncated protein
What is the molecular basis of a frameshift mutation?
deletion or insertion of nucleotide(s)
If it’s 1 or 2 nucleotides you get a shift
If it’s 3 nucleotides you get an additional or a deleted amino acid
Changes the mRNA
To engineer your favorite protein, you are creating point mutations and targeting one base pair. What type of mutation would you do to change the codon and keep the same amino acid?
A. Silent mutation
B. Missense mutation
C. Nonsense mutation
D. Deletion
E. Insertion
A. Silent Mutation
What is a mutation reversion?
a second mutation that can reverse that effect of a mutation.
It fixes the previous mutation.
When a mutation occurs, the result is a mutant. What is the result of a reversion?
a revertant
What is a true revertant?
when the reversion happens at the same site as the original mutation. Results in a wild type genotype
When a reversion occurs at a second site (not the original site) what are the possible results?
Suppressor mutation
- it compensates for the original mutation
Suppressor tRNA
- if you have a stop codon, the mutation happens in the tRNA gene = fixes the nonsense mutation
A second round of mutation was done to restore the phenotype to wild type. The sequencing data revealed that a new mutation restores the original phenotype of your gene. What type of mutant do you have?
A. true revertant
B. revertant
C. suppressor
D. suppressor tRNA
A. true revertant
By which methods do bacteria get new genes?
Transformation
Transduction
Conjugation
What is transformation?
Genetic transfer of free DNA
- from cell lysis or small DNA fragments
Competent cells
- able to take up DNA
Natural Transformation
- happens in nature
DNA uptake
- from some species pili
What is Conjugation?
Genetic transfer requiring cell-cell contact
- plasmid encoded or conjugating pili
Donor (plasmid +) and Recipient (plasmid -)
Fertility (F-) and Plasmid (F+)
-tra region - conjugative pili + type IV secretion system
How does conjugation happen?
You do not need to remember these steps.
What is transduction?
Phages can pick up host DNA and transfer it to a new host
(viral infections)
What is generalized transduction vs specialized transduction?
Generalized Transduction
-lytic phage
-packaging of host DNA
-defective phage
Specialized Transduction
-temperate phages (lysogen/lysogeny)
-host DNA near site of insertion is excised
You need to introduce new genetic material into your favorite bacterium. However, your bacterium is resistant to phage infection and is F+ what methods can you use?
A. conjugation
B. Transduction
C. Transformation
D. All of the above
C. Transformation
What is phage conversion?
Alteration of phenotype by prophages
Gene in the phage genome might confer other functions in the host
Harmless bacteria can become pathogens
ex. Vibrio cholera
-(Prophage + = pathogenic |Prophage - = harmless)
How do prokaryotes defend against phages?
Mutation in receptors
Restriction enzymes
Phage exclusion
Programmed cell death
CRISPR
How do prokaryotes defend themselves from phages by mutations in the receptors?
The phage needs to bind to a receptor. If there is a mutation on the receptor it’s not able to infect that bacterial cell
How do prokaryotes defend themselves from phages by restriction enzymes?
Endonucleases
- specfic sequence
Host protected by methylation
Molecular biology
How do prokaryotes defend themselves from phages by phage exclusion?
Modified DNA, prevent replication
How do prokaryotes defend themselves from phages by programmed cell death?
suicide initiated by toxin-antitoxin system
What is CRISPR?
Cluster Regularly Interspaced Short Palindromic Repeats
Seek and destroy foreign nucleic acid
Adaptive immunity - resistance based on previous infection
- Repeat host sequence
- Spacer - memory of previous invation
- Cas Protein - endonuclease that degrades foreign DNA & integrate new spacers
Your favorite bacterium is sensitive to phages, what protective mechanism could you introduce in your favorite bacterium?
- Infect your bacterium with a defective phage to create a prophage
- Introduce mutations in the phage receptor
- Add a restriction enzyme and a DNA methyltransferase
- Engineer a programmed cell death controlled by a toxin-antitoxin system
- Introduce a CRISPR-cas system
When and why would you induce reversion/suppressor mutations to study bacterial physiology?
redundancies in pathways
metabolic shunt (bypass, alternative routes)
Antibiotic resistance mechanism
What is evolution?
Change in heritable characteristics (ie. DNA) in a population (over time)
Which statement about evolution is true?
A. Evolution is a slow process that improves an individual
B. Evolution is not currently happening in humans
C. Evolution occurs at the population level not at an individual level
D. All mutations are harmful
C. Evolution occurs at the population level not at an individual level
What do you need for evolution to occur?
Change in the genome: mutations, acquisition of new genetic material or genome rearrangement (deletion or duplication)
A process that changes the frequency
- neutral (genetic drift)
- selection (population reduction and “fittest” individuals survive
- bottle-neck (non-selective population decrease)
- founders effect
- co-evolution
Which of the following changes the genotype but will have no effect on the phenotype?
A. silent mutation
B. non-sense mutation
C. missense mutation
D. a 2 base pair deletion
A. Silent mutation
What are recombination events?
events where genes get swapped or rearranged or duplicated or deleted
Enzymes & Homologous recombination
Rearrangement: change of location
ex. 12345 -> 23145
ex. phase variation
ex. antigenic variation
Deletion - can be deleted for no reason
ex. 12345 -> 1345
Duplication - you get a new copy of the gene
ex. 12345 -> 112345
What does homologue mean?
Genes share a common ancestor or origin
What does paralogue mean?
Genes share a common ancestor but have different function
What does orthologue mean?
Genes share a common ancestor and have the same function
What does analogue mean?
No common ancestor or origin but similar function
What is an example of an analogue feature?
wings on birds vs wings on bats
Similarities can arise due to:
- convergent evolution (specific activity)
- gene fusion - partial homology
You are analyzing the sequence and phenotype of two different bacterial species and found a gene with a similar function but not sequence similarity you have?
A. a homologue
B. an orthologue
C. a paralogue
D. an analogue
D. an analogue
Horizontal gene transfer is mediated by:
A. Phages
B. Natural transformation
C. Conjugation
D. All of the above
D. all of the above
How do bacterial cells get new genes?
Conjugation
-bacterial sex
Transduction
- mediated by phages
- phage conversion
Transformation
-Picking DNA up from the environment
What is a genomic island?
a nucleotide sequence that looks completely different than the rest of the genome
How are genomic islands acquired?
Horizontally acquired gene cluster
-different GC content
-different codon usage (codon bias)
-evidence for being part of mobile DNA elements
- may or may not be mobilizable
What is a pathogenicity island?
genome cluster that plays a role in pathogenicity that is absent in non-pathogenic members
Genomic plasticity in bacteria
Genomes are alterable
Fluid exchange of DNA
Rapid adaptation
Survival
Genomic plasticity increases the genetic diversity of the species
What is meant by pangenome?
Collection of genes in 1 species
What is meant by core genome?
genes that are present in all members of the species
typically the genes that are essential for function
What is meant by accessory genome?
genes that are not present in all members of a species.
Expansion of the accessory genomes is mediated by:
A. Mutation
B. Horizontal Gene Transfer
C. Vertical Gene Transfer
D. Genome Rearrangement
B. Horizontal Gene Transfer
What are the co-evolution Hypotheses for the competition modelz?
Red-Queen hypothesis
Evolutionary “arms-race”
Court jester hypothesis
What are the co-evolution hypotheses for the cooperation models?
Green-Beard Effect
Black-queen hypothesis
Which statement about evolution is true?
A. Evolutionary mechanism serve a purpose or stive for perfection
B. Natural selection and genetic drift require variation, which arises from mutation and gene flow
C. “Fitness” refers to the strength, size, or speed of an individual
D. Natural selection and evolution are the same thing
B. Natural selection and genetic drift require variation, which arises from mutation and gene flow
how long does it take for 1 generation of bacteria?
between 10 mins to 24 hours
Which of the following is an application of biotechnologies?
A. Artificial selection of domesticated plants
B. Transgenic plants
C. Bt insecticides (Thuricide)
D. Production of insulin
E. All of the above
E. All of the above
What is biotechnology?
The science of using living systems to benefit humankind
genetic engineering
What are some examples of humans using biotechnology to their advantage?
Cheese, bread, wine, beer, yogurt
plant and animal domestication and artificial selection
transgenic plants
organic and biological insecticides
bio-fertilization
genetic diagnosis and gene therapy
production of vaccines, antibiotics, and hormones
What is recombinant DNA?
the artificial recombination of DNA from two organisms
genetic engineering
- the creation of DNA molecules in the lab by breaking pieces and creating a brand new piece of DNA that would normally not be visible or seen in nature
DNA molecules formed in the laboratory
- genetic recombination
- molecular cloning
Glue genetic material from multiple sources
- creating sequences
To produce insulin using E. Coli, you would need:
A. cDNA of the insulin gene
B. an expression vector
C. Ligase
D. All of the above
D. All of the above
How do we use recombinant DNA?
Get a copy of the gene and put it into a plasmid to obtain a recombinant piece of DNA that we put into E. Coli to eventually produce insulin that we can use for treatment
What is a cloning plasmid?
small pieces of typically circular, double-stranded DNA that replicate independently of the bacterial chromosome
used as vectors to carry DNA fragments
What components make up a cloning plasmid?
Multicloning site
- sequence recognized by a restriction enzyme
Selection marker
- antibiotic resistance
Reporter gene
- lacZ - blue-white screening
What is the role of the restriction enzyme?
restriction endonucleases
restriction enzyme cuts DNA at a characteristic recognition site, a specific, usually palindromic, DNA sequence typically between four to six base pairs in length.
A palindrome is a sequence of letters that reads the same forward as backward.
What is the role of the reporter gene?
blue-white screening
most common reporter gene used in plasmid vectors is the bacterial lacZ gene encoding beta-galactosidase
Blue colonies have a functional beta-galactosidase enzyme because the lacZ gene is uninterrupted, with no foreign DNA inserted into the polylinker site.
white colonies lack a functional beta-galactosidase enzyme, indicating the insertion of foreign DNA within the polylinker site of the plasmid vector, thus disrupting the lacZ gene.
What is an expression plasmid?
Used to express gene and produce protein
Contains a:
Multicloning site
- sequence recognized by restriction enzyme
Selection marker
- Antibiotic resistance
An inducible promoter
- strong repression
A tag for purification
- allows you to fish out that protein using column chromatography or affinity purification
What would you need to replicate DNA in a test tube?
A. Ligase
B. Primers
C. DNA Polymerase
D. Helicase
E. Topoisomerase
F. DNA Polymerase & Primers
F. DNA Polymerase & Primers
How do we amplify DNA?
Dentauring
Reverse transcriptase PCR
- enzyme from viruses
- 5’ to 3’
Annealing
Random DNA primer & dNTPs
Extension
Complimentary DNA (cDNA)
What is the 1st step in amplifying DNA?
always denaturing at 95 degrees C
You need to break and pull the strands apart.
Use a thermal stable polymerase to isolate it from the thermophiles
What happens after you denature the DNA in amplification?
Annealing at -50 degrees C
Need 2 primers that will fix in a specific area in the sequence
reverse complementary to the DNA sequence
What happens after denaturing and annealing in DNA amplification?
Extension at 72 degrees C
Polymerase will bind and initiate the synthesis of a complementary DNA strand resulting in 2 copies of DNA
What enzyme(s) could you use to cut and give DNA in a test tube?
A. DNA Polymerase
B. Ligase
C. Restriction Enzyme
D. Restriction Enzyme & Ligase
E. Helicase
D. Restriction Enzymes & Ligase
Restriction enzyme cuts DNA. It is there as a defense mechanism. Ligase is there to include 2 fragments during the DNA replication step
How can we induce precise mutations?
Site-Directed mutagenesis
- PCR Variant
CRISPR gene editing
How do we cut DNA and glue DNA?
Sticky End Cutting
Blunt End Cutting
How does sticky end cutting work?
A restriction enzyme will recognize a palindrome. an exonuclease will cut the DNA so it has overhangs that are complementary.
How does blunt end cutting work?
A restriction enzyme will recognize a palindrome. An endonuclease will cut the DNA so it has blunt ends.
Blunt ends attach together less efficiently than sticky ends due to the lack of complementary overhangs facilitating the process.
What is the net charge of DNA?
A. Positive
B. Negative
C. Neutral
D. pH-dependent (pka value)
B. Negative
What the benefit to DNA having a negative charge?
We can make it migrate in a mix of agarose or any other polymer without modifying it.
What is agarose?
a purified version of agar?
What is gel electrophoresis?
A process that allows us to visualize DNA
- load the sample
- Add an electric field
- DNA moves based on size. Bulkier fragments take longer to migrate - Add an agent that is fluorescent. It binds to DNA and then fluoresces when viewed under UV light.
What are DNA probes?
Probes can be used to identify different bacterial species in the environment and many DNA probes are now available to detect pathogens clinically.
the DNA probe must be labeled with a molecular tag or beacon, such as a radioactive phosphorus atom or a fluorescent dye so that the probe and the DNA it binds to can be seen
What is the solid blot method of visualizing DNA?
Divide and separate the DNA fragments and see if there is a specific sequence in the sample
Separate by gel electrophoresis, blot it onto a nylon membrane and then add a probe to see if the sequence is present.
what are microarrays?
Microarray analysis is useful for the comparison of gene-expression patterns between different cell types.
Typically, DNA or cDNA from an experimental sample is deposited on a glass slide alongside known DNA sequences.
Once deposited on the slide, genomic DNA or mRNA can be isolated from the two samples for comparison.
Then the two samples of genomic DNA or cDNA are labeled with different fluorescent dyes (typically red and green)
How do bacterial cells acquire new DNA molecules?
A.Transduction (viruses)
B. Natural competence
C. Conjugation (mating)
D. Artificial Competence
E. Natural and artificial competence
F. Viruses, Competence and mating
F. Viruses, competence and mating
How do we transform bacterial cells?
In the lab
Chemically competent or electrocompetent bacteria.
How do we transform eukaryotic cells using electricity?
Make them electro competent.
How do we transform eukaryotic cells using a gold gun?
inject directly using gold particles
How do we transform plants?
use bacteria to transform plants
bacteria will transfer DNA directly into the plant and modify it.
What do white colonies represent?
blue-white screening
A. cells that contain empty vector/plasmid (uncut or self-ligation)
B. Cells that contain a vector/plasmid with an insert in the MCS
C. Cells that have no plasmid/vector
D. Cells with a functional lacZ gene
B. Cells that contain a vector/plasmid with an insert in the MCS
White colony doesn’t have a functional lacZ. The lacZ is the reporter gene that will cleave a lactose analogue that will give you a blue color. So if it is lacZ that is not functional there’s no blue color. If it is white it does have a plasmid if you grow it.
Blue colony = empty vector plasmid or a functional lacZ
Why would you add an antibiotic such as ampicillin into your agar medium when cloning a gene into E. Coli?
A. To prevent the growth of contaminants
B. To prevent the growth of E. Coli that do not have the plasmid
C. To select for E. Coli that have the plasmid with the correct gene
D. To select for E. Coli with a plasmid and prevent growth of E. Coli without the plasmid
D. To select for E. Coli with a plasmid and prevent growth of E. Coli without the plasmid
