Chapter 5 Flashcards
When was Pneumonia frequently fatal and studied by scientists?
1920s
What bacterium was studied in relation to pneumonia in the 1920s?
Streptococcus pneumoniae (pneumococci).
Who discovered two forms of Streptococcus pneumoniae?
Frederick Griffith.
What are the two forms of Streptococcus pneumoniae?
The smooth (S) strain and the rough (R) strain.
What distinguishes the smooth (S) strain from the rough (R) strain?
The S strain has a polysaccharide capsule, while the R strain does not.
What term did Griffith use to describe the new encapsulated bacteria?
“Transformed.”
Who discovered the “transforming principle” and when?
Avery, MacLeod, and McCarty; 1940s
What was identified as the “transforming principle”?
DNA from the dead S strain.
What genetic transfer process was discovered in 1946?
Bacterial conjugation.
Who discovered bacterial conjugation?
Lederberg and Tatum.
When was transduction found?
1951
Who discovered transduction?
Lederberg and Zinder.
What bacterium did Lederberg and Zinder study for transduction?
Salmonella.
What key discovery did Lederberg and Zinder make about gene transfer?
Bacteria did not need physical contact to transfer genetic material.
What did Lederberg and Zinder’s discovery suggest about bacterial gene transfer?
Bacteria do not build a bridge to transfer genes.
What was responsible for DNA transfer in transduction?
Bacteriophages (viruses that infect bacteria).
What did scientists discover while investigating bacterial conjugation?
Plasmids.
Who independently discovered plasmids?
Hayes and Lederberg.
Who proposed the name “plasmids” and in what year?
Lederberg in 1952.
Who proposed plasmid DNA was circular and when?
Allan Campbell; 1962
What was discovered about some Shigella strains?
They carried plasmids that conferred antibiotic resistance.
What could antibiotic-resistant Shigella plasmids do?
Transfer resistance to non-drug-resistant strains.
Who proposed the use of restriction enzymes and plasmids for recombining DNA?
Peter Lobban.
What major breakthrough in genetic engineering was published in 1973?
The artificial construction of a biologically functional plasmid.
Who published the paper on recombinant DNA technology?
Stanley Cohen, Annie Chang, Herbert Boyer, and Robert Helling.
When was the paper of recombinant technology made?
1973
What did the 1973 paper mark the beginning of?
The age of recombinant DNA technology.
What are plasmid maps used for?
To represent plasmids and their functional components.
What is the ori in a plasmid?
A recognition site for DNA polymerase.
What does the ori allow in a plasmid?
Replication and cloning as the bacterium divides.
What is copy number in plasmids?
The number of plasmids per bacterial cell.
What determines the copy number of plasmids?
The ori (origin of replication).
What are β-lactamases?
Enzymes that break down β-lactam antibiotics.
What types of antibiotics do β-lactamases break down?
Antibiotics with a β-lactam ring, such as penicillin and ampicillin.
How do RNA polymerases function on opposite DNA strands?
They read in opposite directions.
What must a plasmid have to express a gene?
A promoter sequence before the coding region.
What is a promoter?
A DNA sequence that provides a landing site for RNA polymerase.
What does a promoter allow RNA polymerase to do?
Transcribe the gene so that the protein can be made.
What are the two main functions of plasmids?
To express recombinant proteins and to carry cloned genes.
What is an expression plasmid?
A plasmid used to express recombinant proteins.
What is a cloning plasmid?
A plasmid used to house and replicate genes.
Why is a cloning plasmid useful?
It is stable and allows millions of copies to be made in bacteria.
Why is DNA manipulation easier in a plasmid?
Plasmids are small, stable, and easily replicated.
What must be done before inserting a gene into a plasmid?
The gene must be cloned.
What method was traditionally used to clone genes into plasmids?
Restriction enzymes.
What method is now used to clone genes into plasmids?
PCR (Polymerase Chain Reaction).
How are genes sometimes cloned into plasmids?
In pieces using multiple steps and intermediates.
What is the multiple cloning site (MCS) in a plasmid?
A string of unique restriction enzyme recognition sites.
What is the function of the MCS in a plasmid?
It allows insertion of the gene of interest.
Why are there many restriction sites in the MCS?
To match the sticky ends of different DNA fragments.
What type of plasmid places the MCS downstream of a promoter?
An expression plasmid.
The protein-coding sequence needs to be cloned in frame for proper expression.
Cloning in frame ensures correct translation of the gene into protein.
When is cloning in frame not important?
It is not important if the gene is only being housed in a cloning plasmid and not expressed.
What are constitutive genes?
Genes that are always expressed.
What are facultative genes?
Genes transcribed only when needed.
How do bacteria regulate some facultative genes?
By using operons.
What is an operon?
A bacterial DNA control unit consisting of one promoter, multiple genes, and a single terminator.
How many mRNA molecules does an operon produce?
One mRNA molecule encoding multiple proteins.
Why are operons beneficial to bacteria?
They allow simultaneous production of multiple proteins for fast adaptation to new food sources.
Do prokaryotes or eukaryotes have operons?
Only prokaryotes have operons.
Who discovered the lac operon?
François Jacob and Jacques Monod
When was the lac operon discovered?
1961
What does the lac operon control?
The production of three enzymes.
What does the araBAD operon control?
Enzyme production for arabinose metabolism.
How are operons controlled?
By repressor proteins encoded by additional genes.
What do repressor proteins bind to?
The operator.
How is an operon turned on?
The sugar (inducer) binds to the repressor, allowing transcription.
What is an inducer?
A sugar molecule that binds to the repressor to turn on an operon.
What is an inducible operon?
An operon that turns on genes that are normally off.
What is a repressible operon?
An operon that turns off genes that are normally on.
What is pBAD18?
A plasmid with the araBAD promoter and the AraC repressor protein gene.
How was GFP expression regulated in pBAD18?
GFP was cloned downstream of the araBAD promoter, making expression arabinose-dependent.
What are shuttle plasmids?
Plasmids that function in both prokaryotes and eukaryotes.
What promoter is used if a gene is expressed in yeast?
A yeast promoter.
What components must a mammalian shuttle plasmid have for propagation in E. coli?
An ori and a bla gene.
What components must a mammalian shuttle plasmid have for expression in mammalian cells?
A selectable marker and a mammalian viral promoter.
Why are viral promoters used in mammalian shuttle plasmids?
Viruses evolved to efficiently use host cell resources.
What sequence do mammalian shuttle plasmids contain to add a poly-A tail?
A polyadenylation sequence.
What is the Ti plasmid?
A naturally occurring tumor-inducing plasmid in plants.
Which bacterium carries the Ti plasmid?
Agrobacterium tumefaciens.
What disease does Agrobacterium tumefaciens cause?
Crown gall disease.
What happens to T-DNA in a Ti plasmid infection?
It integrates into the plant’s genomic DNA.
How is the Ti plasmid modified in the lab?
Tumor-inducing genes are removed, and foreign genes are inserted into the T-DNA.
How is the modified Ti plasmid propagated?
In A. tumefaciens using its natural regulatory sequences.
What drives gene expression in engineered Ti plasmids?
Plant viral promoters.
What sequences are required in engineered Ti plasmids?
Terminator sequences, often cloned from natural Ti plasmids.
What is transformation?
The process of introducing foreign DNA into a cell.
How is transformation efficiency measured?
Number of transformed bacteria per microgram (μg) of DNA.
What is calcium chloride transformation?
A method where actively dividing bacteria are washed in ice-cold calcium chloride.
What does calcium chloride do to bacterial cells?
Calcium chloride makes bacterial cells chemically competent and more permeable to DNA.
How are chemically competent bacteria transformed?
Plasmid DNA is added to the cells, incubated on ice, heat shocked at 42°C for 50 seconds, then placed back on ice.
What is added to transformed cells before incubation at 37°C?
Non-selective growth medium is added to allow recovery.
How does electroporation transform cells?
Electricity disrupts cell membranes, temporarily increasing permeability.
What is transfection?
Transfection uses lipid vesicles to deliver plasmid DNA into eukaryotic cells.
What method is used to penetrate plant cell walls for transformation?
Biolistics, which shoots DNA-coated gold or tungsten particles into cells under helium pressure.
Besides plants, what other cells can be transformed using biolistics?
Biolistics can also be used to transform other eukaryotic cells.
How can genes be introduced into plants using bacteria?
A genetically modified Ti plasmid from Agrobacterium tumefaciens integrates foreign genes into the plant genome.
How do scientists introduce Ti plasmids into plant cells?
By infecting plant cells with Agrobacterium cultures containing engineered Ti plasmids or using biolistics.
What is transduction?
Transduction is the use of bacteriophages to deliver genes into bacterial cells.
What viruses are commonly engineered to express recombinant genes?
Baculoviruses, which naturally infect insect larval cells.
How do retroviruses integrate foreign genes into mammalian cells?
They convert their RNA genome into DNA and insert it into the host genome.
What is selection in genetic transformation?
Selection is the process of identifying transformed cells.
How are new plasmids typically generated?
By ligating a new DNA fragment into the multiple cloning site (MCS) of an existing plasmid backbone.
What is the purpose of selection systems in plasmid transformation?
To distinguish plasmids with new DNA inserts from recircularized plasmids without inserts.
What gene does the pUC18 plasmid contain for selection?
The lacZ gene, which encodes β-galactosidase.
What happens when β-galactosidase cleaves X-gal?
X-gal turns blue, causing bacterial colonies with plasmids to appear blue.
What color do bacterial colonies appear when the lacZ gene is disrupted by an insert?
White, because β-galactosidase is not produced.
How have scientists engineered the lacZ gene for selection?
A multiple cloning site (MCS) is inserted into lacZ without disrupting its function unless a new DNA fragment is ligated.
What is positive selection in transformation?
A system where unsuccessful ligations cannot grow due to toxic MCS insertion.
What factors affect transformation efficiency?
Bacterial growth phase, plasmid amount and supercoiling, bacterial type, heat shock temperatures, and transformation method.
Which transformation method is more efficient, electroporation or calcium chloride treatment?
Electroporation is usually more efficient.
Is plasmid transformation more efficient than ligation transformation?
Yes, plasmid transformation is much more efficient.
What bacterium is most commonly used to produce plasmids?
Escherichia coli (E. coli).
What is a miniprep?
A process of growing a bacterial culture and purifying the plasmid DNA.
What are the two purposes of a miniprep?
1) To purify plasmid DNA from bacteria and separate it from genomic DNA. 2) To amplify plasmid DNA for further experiments.
What are the three key requirements for optimal E. coli growth?
Nutrients, oxygen, and a warm temperature.
What are the four phases of a bacterial growth curve?
Lag, growth, stationary, death.
How are transformed bacteria harvested after overnight growth?
By centrifugation, followed by plasmid DNA purification.
What happens when bacteria are spun in a microcentrifuge?
The bacteria form a dense pellet at the bottom of the tube.
How is the bacterial pellet prepared after centrifugation?
It is resuspended in a buffered solution.
What must be done to bacteria to extract plasmid DNA?
They must be lysed to break the cell wall and membrane.
What chemicals are used to lyse bacterial cells?
Sodium hydroxide, SDS, and EDTA.
What role does EDTA play in bacterial lysis?
It removes magnesium ions, destabilizes the cell wall, and inactivates DNases.
What happens when the bacterial cell wall is weakened?
The cell bursts, and alkaline conditions break hydrogen bonds in DNA.
How is the alkaline pH neutralized in plasmid extraction?
A low pH, high salt solution is used.
What effect does high salt concentration have in plasmid extraction?
It causes proteins to precipitate and gDNA to aggregate.
Why do plasmids remain soluble while gDNA aggregates?
Their single strands remain linked and reanneal into double-stranded molecules.
How is cellular debris removed after lysis?
By centrifugation, leaving plasmid DNA in the supernatant.
What is the final step in plasmid DNA purification?
The purified plasmid DNA is collected from the supernatant.
What is neutralized in plasmid extraction?
A low pH, high salt solution is used.
What is the final step in plasmid DNA purification?
Removal of salts and contaminants.
What was historically used to desalt and concentrate DNA?
Alcohol precipitation.
What are the steps of alcohol precipitation?
Add alcohol, incubate, centrifuge, wash, centrifuge again, air dry, and hydrate.
What modern method is commonly used for plasmid purification?
Chromatography columns.
How does DNA bind to chromatography columns?
It binds strongly to silica in high salt conditions.
How is DNA eluted from a chromatography column?
Using a low-salt elution solution.
What principle does gel quantitation rely on?
The intensity of a DNA band is proportional to DNA amount.
What are mass rulers used for in gel quantitation?
To compare DNA concentrations.
How does linear DNA migrate in agarose gel electrophoresis?
Proportionally to its size.
How does circular DNA migrate in agarose gel electrophoresis?
Differently than linear DNA.
What should be used for plasmid quantitation in gel analysis?
The most intense plasmid band.
What type of light source is required for spectrophotometric DNA quantitation?
Ultraviolet (UV) light.
What is the absorbance of a 50 μg/ml double-stranded DNA solution at 260 nm?
1 (A260 = 1).
If A260 is 0.5, what is the DNA concentration?
25 μg/ml.
Why are quartz cuvettes preferred for UV spectrophotometry?
They allow UV light transmission.
Why are plastic resin cuvettes used instead of quartz?
They are cheaper and less fragile.
What are micro cuvettes used for?
To conserve DNA samples.
How is DNA typically diluted before spectrophotometric analysis?
10 to 100 times.
How do single-stranded DNA and RNA absorb UV light compared to double-stranded DNA?
Differently; 33 μg/ml ssDNA and 40 μg/ml RNA both have A260 of 1.
How can spectrophotometers assess DNA purity?
By measuring the A260/A280 ratio.
What absorbs light at 280 nm in spectrophotometry?
Proteins (due to aromatic rings).
What absorbs light at 260 nm in spectrophotometry?
DNA.
What does a spectrophotometer’s automatic setting measure in DNA purity assessment?
The A260/A280 ratio.
What does an A260:A280 ratio <1.7 indicate?
Protein contamination.
What must be done if the A260:A280 ratio is well below 1.7?
The DNA must be purified before proceeding.
What is a NanoDrop spectrophotometer?
A small-volume spectrophotometer that measures samples without cuvettes.
What measurements can a NanoDrop spectrophotometer provide?
A260 and A260:A280 ratio.
What is a fluorometer used for?
Measuring DNA at very low concentrations.
What type of DNA does a fluorometer specifically measure?
Double-stranded DNA.
Why is a fluorometer useful for contaminated DNA samples?
It can specifically measure double-stranded DNA even if RNA is present.
