exam 2- potential questions

Given a "recipe" for a specific culture medium, identify a complex or defined medium.

A defined medium contains only known quantities of specific, chemically defined compounds (e.g., glucose, salts, amino acids). A complex medium contains undefined ingredients, like extracts or digests of plant or animal tissues, which provide a variety of nutrients in unknown quantities (e.g., nutrient broth, yeast extract).

Which enzyme catalyzes the reaction: H₂O₂ + 2H+ → 2H₂O

catalase

In a standard growth curve, what phase represents when the number of cells growing and dying are about equal.

stationary phase

the growth rate equals the death rate due to limited nutrients or the accumulation of waste products

In a figure/graph, which line best depicts a psychrotroph incubated at 0°C?

b

A media that has a pH indicator and high salt concentration can likely _____ the growth of most bacteria.

inhibit the growth of most bacteria, as the high salt concentration can be selective (e.g., for halophiles) and the pH indicator can show a shift in pH that indicates metabolic activity or inhibition.

Given a list, be able to distinguish which is NOT a direct measurement of microbial growth.

Direct measurements of microbial growth include counting colony-forming units (CFUs), measuring cell mass (dry weight), or using a microscope to count cells. A spectrophotometer measuring turbidity is an indirect method, as it estimates growth based on light scattering, not directly counting the cells

If 100 colonies are on a 1:1000 dilution plate, how many bacteria/mL were in the original sample?

100 colonies × 1000 = 100,000 bacteria/mL in the original sample

A sample of ocean water is tested for its bacterial content in a plate count assay. A one-milliliter sample of the water is diluted in a 1:10 dilution series. 0.1 milliliter of the second dilution tube is plated in a pour plate. After incubation, the plate has 191 colonies, indicating that the original water sample contained

191,000 bacteria/mL in the original water sample.

Process of destroying pathogenic microorganism from skin or living tissue is called:

Describes the pattern of microbial death?

Antisepsis. This refers to the process of killing or inhibiting the growth of pathogens on living tissues, such as skin.

Given a known microbial death curve (like Fig 7.1.) If you have 1,000,000 cells, how many survivors would there be after x min?

Microbial death typically follows a logarithmic pattern, meaning that the number of surviving microorganisms decreases exponentially over time when exposed to a lethal agent (e.g., heat, disinfectants).

A suspension of 106 Bacillus cereus endospores was put in a hot-air oven at 170°C. Plate counts were used to determine the number of endospores surviving at the time intervals shown. In a given figure/graph, what is the thermal death time?

The thermal death time (TDT) is the time required to kill a specific microorganism at a given temperature. From the graph, you would identify the time at which the number of surviving endospores drops to zero or becomes negligible at 170°C.

In a particular liquid culture, 100% of the microbes are killed by 12 minutes at 30 degrees C. This is called:

thermal death point (TDP), which is the minimum time and temperature combination required to kill all microorganisms in a given culture

Which of the following treatments from a given list is the most effective for controlling microbial growth?

generally, autoclaving (using steam under pressure) or sterilizing filters are highly effective for controlling microbial growth. High concentrations of disinfectants or broad-spectrum antibiotics may also be very effective depending on the microorganism.

Which of the following disinfectants from a given list acts by disrupting the plasma membrane?

Disinfectants like phenols and alcohols (e.g., ethanol and isopropanol) act by disrupting the plasma membrane, causing leakage of cellular contents and eventual cell death

How does osmotic pressure provide microbial growth control?

Osmotic pressure can control microbial growth by creating a hypertonic environment. This causes water to leave microbial cells, leading to dehydration and plasmolysis (shrinking of the cell membrane), which inhibits the growth or kills the microorganism.

Which chemical methods disrupt the plasma membrane? Denature proteins? Damages DNA?

Disrupting the plasma membrane: Alcohols (e.g., ethanol, isopropanol) and phenolic compounds.

Denature proteins: Heat (like autoclaving), heavy metals (e.g., silver, mercury), and halogens (e.g., chlorine, iodine).

Damages DNA: Ultraviolet (UV) radiation and certain chemicals like formaldehyde and ethylene oxide can damage DNA, leading to mutations or death.

Repeating sequences of noncoding DNA that are used in forensic analysis is called

Short Tandem Repeats (STRs)

The antibiotic chloramphenicol binds the 50S large subunit of a ribosome. From this information you can conclude that chloramphenicol

it inhibits protein synthesis by preventing peptide bond formation, thus acting as an antibiotic

Give an example of horizontal gene transfer

Conjugation, Transformation, Transduction

How is DNA transferred in transformation from a donor to a recipient cell?

a donor cell releases free DNA into the environment, which is then taken up by a recipient cell, integrating the foreign DNA into the recipient’s genome

If tryptophan is present, the repressible operon is:

turned off, as tryptophan acts as a corepressor, binding to the repressor to stop transcription

How do you identify an auxotroph?

an organism that cannot synthesize a specific compound (e.g., an amino acid or vitamin) and requires it from the environment for growth

If you have an Hfr, Trp+ cell that recombines with an F-, Trp- cell, what might happen to the F-, Trp- cell? (what traits could it acquire?)

The F-, Trp- cell could acquire the Trp+ trait (tryptophan production) through genetic recombination if the donor DNA (Hfr) includes the trp gene

If an F+, Cys+ cell recombines with an F-, Cys- cell, what might happen to the F-, Cys- cell?

The F-, Cys- cell could acquire the Cys+ trait (cysteine synthesis) if the donor DNA (F+) includes the cys gene

What does a vector, like PUC19, contain in order to easily take an insert and transform a cell?

A vector like PUC19 contains the following key features:

Origin of replication (ori): This allows the vector to replicate inside a host cell.

Multiple cloning site (MCS): A short DNA sequence with several restriction enzyme sites, which makes it easy to insert foreign DNA (the insert).

Selectable marker: Often an antibiotic resistance gene (e.g., ampicillin resistance) that allows the selection of cells that have taken up the vector.

Promoter: A sequence that can initiate transcription of the insert in the transformed cell.

Screening marker: In PUC19, it typically has a lacZα gene for blue/white screening of recombinant colonies.

Advantages of a viral vector over a plasmid vector is:

Higher Efficiency: Viral vectors, especially those based on retroviruses or adenoviruses, are more efficient in delivering genetic material into host cells, especially for cells that are difficult to transfect with plasmids.

Incorporation into Host Genome: Some viral vectors, like retroviral vectors, integrate the insert into the host genome, leading to stable expression of the inserted gene.

Larger Insert Capacity: Certain viral vectors can carry larger inserts compared to plasmid vectors, which is useful when working with large genes or genomic sequences.

Use in Eukaryotic Cells: Viral vectors are better suited for gene delivery in eukaryotic cells, including human cells, whereas plasmids are more commonly used in bacteria.

Describe key features of a restriction enzyme

Sequence Specificity: Restriction enzymes recognize specific DNA sequences, often palindromic (e.g., EcoRI recognizes GAATTC).

Endonuclease Activity: They cut DNA at specific sites within or near their recognition sequences.

Types of Cuts: Some enzymes produce sticky ends (overhanging single-stranded ends) while others create blunt ends (straight cuts).

Cofactor Requirement: Most restriction enzymes require magnesium ions (Mg²⁺) as a cofactor to cut the DNA.

Use in Genetic Engineering: They are widely used in cloning, DNA mapping, and gene editing.

Place the steps of constructing a genomic library in order

Isolation of genomic DNA: Extract total DNA from the organism of interest.

Fragmentation of DNA: The genomic DNA is cut into smaller pieces using a restriction enzyme.

Insertion into vector: The fragmented DNA is inserted into a vector (plasmid or bacteriophage).

Transformation or Transfection: The recombinant vectors are introduced into host cells (e.g., bacteria or yeast).

Selection and screening: Host cells are selected (usually using a selectable marker), and colonies are screened to identify those containing the desired DNA fragments.

Storage: The genomic library is stored for future use.

Explain how DNA replication and PCR are similar.

Template-dependent synthesis: Both processes use a DNA template to synthesize a complementary strand.

Polymerase involvement: Both involve DNA polymerase (in replication, it's the DNA polymerase in the cell; in PCR, it's a heat-stable version, like Taq polymerase).

Direction of synthesis: Both processes synthesize DNA in the 5' to 3' direction.

Priming: Both processes require primers to initiate synthesis. In DNA replication, RNA primers are used, while in PCR, short DNA primers are used.

Nucleotide addition: Both processes add nucleotides to the growing DNA strand by complementary base pairing.

Be able to predict the product at every step of PCR and what would happen if a step was skipped or an enzyme or nucleotide was missing

Step 1: Denaturation (95°C): The double-stranded DNA template is denatured into two single strands.

If skipped: The DNA won't separate into single strands, and no amplification will occur.

Step 2: Annealing (50-65°C): The primers bind to their complementary sequences on the single-stranded DNA.

If skipped: No primers will bind, and the polymerase can't begin synthesizing new DNA.

Step 3: Extension (72°C): Taq polymerase extends the primers and synthesizes the new strand by adding nucleotides complementary to the template strand.

If skipped: The DNA will not be amplified, as no new strands are synthesized.

If an enzyme is missing (e.g., Taq polymerase): The reaction will not proceed since no DNA synthesis will occur.

If nucleotides are missing: The polymerase won’t be able to synthesize a full complementary strand, and amplification will be incomplete.

How is CDNA made?

synthesized from mRNA using the enzyme reverse transcriptase. The process involves:

Isolating mRNA from cells.

Using a specific primer (often oligo(dT) or random primers) to bind to the mRNA.

Reverse transcriptase synthesizes a complementary DNA strand to the mRNA.

The mRNA strand is degraded, and a second DNA strand is synthesized to create a double-stranded cDNA molecule.

Which technology uses DNA probes to detect DNA fragments?

Southern blotting: This technique involves transferring DNA fragments from an agarose gel to a membrane and then hybridizing them with a labeled DNA probe to detect specific sequences.

How can genomes from microbes that are not able to grow in the lab still be studied?

Metagenomics: DNA can be extracted directly from environmental samples (e.g., soil, water, or human microbiomes) and sequenced without the need to culture the microbes.

Shotgun Sequencing: The DNA of all microbes in a sample is fragmented and sequenced, and then bioinformatic tools are used to reconstruct the genomes.