Deck 12: Biotechniques and Synthetic Biology

Question 1

Advantages in real-time PCR over regular, end-time PCR include all of the following EXCEPT

A) initial amounts of template DNA can be quantified by the length of time it takes to detect PCR products.
B) amplified DNA is quantified while the PCR is still taking place.
C) earlier increments in fluorescence indicate larger amounts of template DNA in the original sample.
D) real-time PCR uses GadX.
E) real-time PCR can be used to quantify DNA or RNA in a sample.

Answer 1

D

Question 2

The colorless molecule o-nitrophenyl galactoside (ONPG) can be used as an artificial substrate to assay b-galactosidase in cell extracts of Escherichia coli in which lacZ is used as a reporter gene. Which of the following statements is NOT true with respect to the use of ONPG?

A) Hydrolysis of ONPG by b-galactosidase produces yellow o-nitrophenol.
B) o-nitrophenol can be measured with a spectrophotometer.
C) Hydrolysis of ONPG by b-galactosidase also releases colorless galactose.
D) The o-nitrophenol moiety is blue and more suitable for use in solid media.
E) The o-nitrophenol moiety is green and less suitable for use in solid media.

Answer 2

D

Question 3

Acid-resistant Escherichia coli cells counteract H⁺ in their cytoplasm through the combined action of two proteins: a decarboxylase enzyme and

A) H+ channel.
B) [H+]-ATPase.
C) GABA/glutamate antiporter.
D) putative DNA-binding protein.
E) putative antiporter.

Answer 3

C

Question 4

A common approach to study a certain metabolic process is to isolate organisms defective in that particular process. These organisms are called

A) agents.
B) wild type.
C) prototrophs.
D) mutants.
E) vectors.

Question 5

The phenomenon that allows a quencher dye to reduce the fluorescence of the reporter dye in real-time PCR techniques is called

A) nuclear magnetic resonance.
B) fluorescence resonance energy transfer (FRET).
C) atomic force tunneling.
D) magnetic resonance imaging.
E) random nicking.

Question 6

What kind of mutation is produced by transposable elements?

A) inversion
B) point
C) insertion
D) substitution
E) transposition

Question 7

The genes for proteins such as b-galactosidase (lacZ) or green fluorescent protein (gfp) can be fused to the promoter of a gene of interest. Which of the following statements is NOT correct about gfp and lacZ?

A) They are called reporter genes.
B) The enzymatic activity of b-galactosidase can be easily assayed.
C) GFP is visualized by fluorescence microscopy.
D) Both genes are of viral origin.
E) The gfp gene is of eukaryotic origin, whereas lacZ is a bacterial gene.

Question 8

Which of the following is correct regarding operon fusions?

A) They expose both transcriptional and translational controls.
B) Both target and reporter genes have their own promoter sequences.
C) Both target and reporter genes have ribosome-binding sites.
D) Only the reporter gene has a promoter.
E) They produce a single transcript and a single hybrid reporter-target polypeptide.

Question 9

CRISPR-Cas9, a prokaryotic defense mechanism, is a powerful emerging tool in biotechnology for all of the following EXCEPT

A) allowing the editing of defective genes in eukaryotic cells.
B) introducing insertions, deletions, or sequence modifications into a given genome with CRISPR-Cas9.
C) regulating gene expression in bacteria with CRISPRi, in which case Cas9 is no longer active as a nuclease.
D) marking a defective gene with antibiotic resistance.
E) disrupting latent HIV provirus in infected cells.

Question 10

What is the main indication that the figure below depicts a transcriptional (operon) fusion?

A) A single mRNA is transcribed, from which two proteins are translated.
B) The reporter gene keeps its biological properties: in this case, fluorescence.
C) Transcription proceeds from a single promoter.
D) The target gene maintains its ribosomal binding site.
E) The reporter gene is inserted into the target gene in the proper codon-reading frame.

Question 11

What kind of gene expression controls are revealed by operon fusions?

A) transcriptional
B) translational
C) both transcriptional and translational
D) posttranslational
E) pretranscriptional

Question 12

His₆-fusion-tagged proteins can be recovered from affinity chromatography columns because the histidine tag tightly binds to beads coated with ________ ions. The elution buffer contains ________, which has stronger affinity for these ions.

A) copper; sulfate
B) magnesium; EDTA
C) iron; citrate
D) calcium; EGTA
E) nickel; imidazole

Question 13

Annotation of sequences of transposon insertion sites in the chromosome of Escherichia coli O157:H7 cells that had lost their acid-resistance properties revealed four genes: two encoding isoforms of glutamate decarboxylase (GadA and GadB) and one encoding a glutamate/gamma-aminobutyric acid (GABA) antiporter. What does the fourth gene encode?

A) a heat shock protein
B) a possible regulator
C) a glutamate/GABA symporter
D) a GABA uniporter
E) a glutamate carrier protein

Question 14

Sigma B is a stress-induced transcription factor in Bacillus subtilis. Bacteria producing a sigma B-YFP fusion protein were exposed to constant energy stress, and fluorescence pulses emitted by individual cells were detected by time-lapse microscopy. Could this result have been obtained using a sigma B-b-gal fusion protein?

A) Yes. Hydrolysis of X-Gal by b-galactosidase produces a blue product detectable by eye.
B) Yes. Hydrolysis of ONPG by b-galactosidase in liquid medium produces an orange compound measurable by spectrophotometry.
C) Yes. It depends on how well the reporter gene is inserted into the vector with respect to the position of the ribosome-binding site.
D) No. Whereas YFP fluorescence can be carefully observed and measured by time-lapse microscopy, b-gal activity is measured as an average of the population as a whole.
E) No; the product would not be detectable by eye.

Question 15

What type of reporter fusions can expose both transcriptional and translational controls?

A) operon fusions
B) gene fusions
C) either operon fusions or gene fusions
D) a fluorescent protein construct such as gfp, yfp, rfp, and so on
E) lac operon

Question 16

For a reporter protein to be synthesized, its gene must be fused in the proper codon reading frame with respect to the

A) ribosome binding site.
B) promoter of the gene of interest.
C) vector origin of replication.
D) target gene.
E) vector's antibiotic marker sequence.

Question 17

Gene fusions of a protein of interest and GFP, or its derivatives, can be used in all of the following EXCEPT

A) studying protein targeting to organelles in eukaryotic cells.
B) monitoring microbial protein movement inside infected eukaryotic cells.
C) tracking viral protein movement inside or between infected eukaryotic cells.
D) detecting interactions between microbial extracellular proteins and attachment surfaces.
E) CRISPR-Cas9.

Question 18

Which answer choice INCORRECTLY labels the figure below?

A) D indicates the CO₂ entering the cell.
B) C points to GABA.
C) A marks the GABA/glutamate antiporter.
D) D is the label for H+ entering the cell.
E) B points to glutamate.

Question 19

Which answer choice INCORRECTLY labels the figure below?

A) A-DNA primer
B) B-transposon
C) C-amplification of gene-transposon junction
D) D-interface gene-transposon
E) There is no incorrect label.

Question 20

Which probing technique detects DNA immobilized on a membrane using labeled DNA fragments?

A) northern blot
B) southern blot
C) western blot
D) polyacrylamide gel electrophoresis in SDS
E) isoelectrofocusing

Question 21

How is bacterial DNA cross-linked to putative binding proteins for chromatin immunoprecipitation (ChIP)?

A) exposure to UV light
B) treatment with formaldehyde
C) treatment with heat
D) reduction with dithiothreitol
E) reduction with b-mercaptoethanol

Question 22

An advantage of using fluorescent protein technology to track protein movements in cells and tissues is that

A) intracellular GFP-protein fusions can be detected by fluorescence microscopy in vivo.
B) a mobile protein can be tracked as it moves through organs or tissues.
C) a fusion with a mutant protein might reveal incorrect localization or movement.
D) it can identify the gene encoding prey protein.
E) it can be used to detect protein-protein interactions.

Question 23

The applications of the yeast two-hybrid method include all of the following EXCEPT

A) mapping protein-protein interactions in E. coli.
B) identifying of physiologically significant protein-protein interactions as potential antibiotic targets.
C) finding unknown proteins ("prey") that interact with proteins of known functions ("bait").
D) mapping protein-protein interactions in M. tuberculosis.
E) the ability to probe interactions in vivo.

Question 24

Genes coding for insecticidal peptides have been engineered into the genomes of some crops. Which of these peptides are naturally produced by the bacterium?

A) Bacillus thuringiensis
B) Agrobacterium tumefaciens
C) Anabaena variabilis
D) Erwinia carotovora
E) Escherichia coli

Question 25

Double-stranded DNA probes must be denatured when used in northern blot experiments to ensure that

A) they are properly radioactively labeled or tagged with a dye.
B) the DNA coding strand hybridizes to RNA on the membrane.
C) both DNA strands bind the single-stranded RNA on the membrane.
D) the RNA coding strand hybridizes to DNA on the membrane.
E) an RNA strand binds to another RNA strand on the membrane.

Question 26

In a DNA protection assay gel, it is necessary to run sequencing ladders of the putative protein-binding DNA region along with DNase I-treated samples in order to

A) establish the position of the binding protein relative to the transcription start site.
B) provide molecular weight markers.
C) check that the gel running conditions are correct.
D) check for nonspecific DNA degradation.
E) check for specific DNA degradation.

Question 27

The purpose of a DNA protection assay is to determine what nucleotide sequences directly interact with which macromolecules?

A) DNA-binding proteins
B) rRNA
C) endonucleases
D) exonucleases
E) tRNA

Question 28

In a yeast two-hybrid analysis of several strains containing different prey/bait combinations, the screening of interacting proteins is done by growing cells on a medium containing X-Gal. White and blue colonies are observed after incubation. Which of the following statements is NOT true regarding blue/white selection in the yeast two-hybrid method?

A) Blue colonies result from X-Gal hydrolyzed by the reporter enzyme b-galactosidase.
B) White colonies result from cells in which the prey and bait proteins do not interact.
C) Galactose released from X-Gal hydrolysis does not contribute to a colony's blue color.
D) White colonies indicate that b-galactosidase catalyzed the hydrolysis of X-Gal.
E) Blue colonies are an indication that GAL4 activated transcription of GAL1-lacZ.

Question 29

In the first stage of the western blot procedure, a technique known as SDS-PAGE is used to separate proteins according to their

A) isoelectric points.
B) quaternary structures.
C) molecular weights.
D) amino acid sequences.
E) secondary structure compositions.

Question 30

Which technique is used to select novel enzymes or peptides produced through directed evolution?

A) mutagenesis with transposons
B) enzyme phage display
C) multiplex PCR
D) ethane methyl sulfonate mutagenesis
E) directional cloning

Question 31

Bacillus thuringiensis stores insecticidal proteins

A) surrounding the spore.
B) in the cytoplasm.
C) as proteins crystallized in the parasporal body.
D) as part of the spore.
E) in the plasma membrane.

Question 32

The figure below shows a gel with Escherichia coli DNA fragments separated by size. The marker lanes help to locate the transcription start following the promoter region of a gene encoding for glutamate decarboxylase (GadA). There are five lanes from an experiment in which varying amounts of the protein GadX were added to DNA prior to treatment with DNase I. What do the roman numeral boxes indicate?

A) change RNA to DNA.
B) topoisomerases-binding regions
C) thus not protected
D) translational start
E) GADF-binding site

Question 33

A protein of interest can be specifically detected on western blots; this technique requires all of the following EXCEPT

A) primary antibodies produced against the protein under study.
B) an enzyme-tagged secondary antibody, directed against the primary antibody.
C) an easily detectable product formed by the enzyme in the tagged secondary antibody.
D) labeled DNA.
E) antibodies to detect quantity and size of specific proteins in cell extracts.

Question 34

The uses for phage display include all the following EXCEPT

A) antibody fragments cloned from synthetic sources.
B) the identification of high-affinity peptides that bind to attachment proteins of some eukaryotic viruses and effectively block viral infection.
C) antibody fragments cloned from natural sources.
D) collections of mutant enzymes to be tested for activity.
E) directed evolution projects.

Question 35

DNA protection assays identify specific nucleotide-protein contacts. What is the role of deoxyribonuclease (DNase) I treatment in footprint analysis?

A) DNase I is used to break DNA previously bound to protein.
B) Protein-bound DNA is protected from DNase I and it can be isolated and characterized.
C) DNase I prepares protein-bound DNA for sequencing.
D) DNase I is used to cleave Taq polymerase.
E) DNase I synthesizes DNA from the upstream primer.

Question 36

Real-time PCR can be used to quantify the original amount of RNA in a sample. What additional step must be accomplished in this particular application?

A) the careful design of specific forward and reverse primers
B) RNA must first be converted to cDNA.
C) RNA must be denatured with formaldehyde to avoid secondary structures.
D) the separation of mRNA from other RNA species
E) DNA is converted to RNA.

Question 37

The migration of linear DNA molecules in agarose or polyacrylamide gels occurs at a rate that is

A) independent of their size.
B) directly proportional to their size.
C) inversely proportional to their size.
D) dependent on the AT content.
E) dependent on the GC content.

Question 38

In the yeast two-hybrid analysis technique, in vivo protein-protein interactions are explored. The "bait" and "prey" proteins are translationally fused to the cleaved activation and DNA-binding domains of the GAL4 transcription factor. What is the target gene of GAL4?

A) lacZ
B) gadA/B
C) GAL1
D) lacY
E) lacA

Question 39

Which of the following experimental procedures is NOT used in ChIP-seq?

A) DNA-protein complexes are cross-linked.
B) DNA is extracted and sheared.
C) Bead-bound antibodies to transcription factors are used to precipitate transcription factors cross-linked with DNA.
D) The bead-bound antibodies are precipitated using a secondary antibody.
E) Transcription factors are released from DNA, amplified and identified by sequencing.

Question 40

Potential vaccine antigens made by transgenic plants include

A) foot-and-mouth surface antigen.
B) Norwalk virus glycoprotein.
C) rabies virus glycoprotein.
D) Entertoxin A subunit.
E) Hepatitis A virus.

Question 41

Both southern and northern blots involve the use of agarose gels. How do the types of agarose gels differ and why?

Question 42

Describe the difference between autoradiography and phosphor imaging.

Question 43

How do operon fusions reveal transcriptional control of a target gene?

Question 44

The replacement of the genome of Mycoplasma capricolum with the modified genome of M. mycoides is considered to be a significant accomplishment because it

A) demonstrates that genetic manipulation of recalcitrant organisms is impossible.
B) opens the possibility to redesign prokaryotic genomic systems.
C) enables gene editing of species.
D) merges the genomes into a hybrid.
E) creates a riboswitch board.

Question 45

Use the figure below to describe the model of acid resistance in Escherichia coli O157:H7. Make sure to identify the molecules involved using the letters on the figure.

Question 46

Existing biocontainment strategies employ natural auxotrophies that are not easily met in natural, or conditional suicide switches introduced into the organism's genome. However, these mechanisms can be compromised through

A) genetic mutation.
B) synthetic auxotrophy.
C) synthetic biology.
D) logic gates.
E) genetic circuit.

Question 47

Scientists have taken the genome of the bacterial species ________ and moved it to yeast.

A) Mycobacterium leprosae
B) Mycobacterium tuberculosis
C) Mycoplasma mycoides
D) Escherichia coli
E) Mycoplasma pneuomoniae

Question 48

What is the simplest definition of a genetic "logic gate"?

A) a promoter (which interacts with input signals such as regulatory proteins) and a gene, which produces mRNA and protein (output signal)
B) The interaction among proteins and specific DNA sequences modulate gene expression.
C) the combination of genetic elements from different organisms to engineer a novel metabolic pathway or reproduce one in vivo
D) determine whether a gfp gene is turned on or off
E) bind inducer molecules and have an effect on gene expression

Question 49

In the figure below, the labels for the genetic logic gates are missing. Which answer choice locates them correctly?

A) i) Buffer, ii) NOT, iii) OR
B) i) NOT, ii) Buffer, iii) OR
C) i) Buffer, ii) OR, iii) NOT
D) i) OR, ii) Buffer, iii) NOT
E) i) NOT, ii) OR, iii) Buffer

Question 50

In isolating acid-sensitive Escherichia coli using tetracycline-resistant transposons, why is it necessary to screen first for tetracycline resistance and then for acid sensitivity?

Question 51

The role of kill switches in genetically engineered microorganisms is to

A) serve as a fail-safe mechanism to timely kill the organisms after they have fulfilled their purpose.
B) induce proliferation of reproduction to kill a host cell.
C) destroy organisms that have a risk of biological contamination or invasion.
D) block the translation of CcdB.
E) elimiate toxins within the cell.

Question 52

What are translational fusions used for? How are they constructed?

Question 53

Use the figure below to describe how the CRISPR-Cas9 editing tool is used in eukaryotic cells.

Question 54

A "buffer gate" in synthetic biology can best be described as

A) a single repressor molecule (R1) that prevents the production of an output molecule (Pr1).
B) alternate input molecules (I1 or I2) that drive the production of alternative proteins (Prn).
C) a single input inducer molecule (I1) that leads to the production of a single product (Pr1).
D) kill switches.
E) a potent DNA-damaging toxin.

Question 55

Which of the following is correct regarding biological toggle switches?

A) They can be thought of as two back-to-back OR gates.
B) They can be described as straightforward "buffer gates."
C) They consist of a single NOT gate.
D) Two inducer signals are necessary to toggle the switch.
E) They have two repressor genes whose products can repress each other's transcription.

Question 56

Noise in synthetic biology is a result of the

A) ara operon.
B) lac operon.
C) dual-feedback oscillator.
D) fluctuation of gene expression among single-cells in a population.
E) oscillatory genetic circuit.

Question 57

The type of genes that encode proteins whose activity is easily assayed are

A) deep sequencing genes.
B) transcriptional fusions.
C) operons.
D) reporter genes.
E) translational fusions.

Question 58

How were GadE and GadX demonstrated to be transcription factors for the gadA gene?

Question 59

Potential applications of bacterial oscillator switches include all of the following EXCEPT the

A) study of controlled interactions between two feedback genetic loops.
B) design of bacterial genetic circuits to detect toxins.
C) design of bacterial clocks.
D) reduction of noise in biological circuits.
E) reactivity to concentrations of specific molecules such as arabinose.

Question 60

Which kind of phage vectors are most frequently used in phage display technology?

A) double-stranded DNA T-odd
B) double-stranded DNA T-even
C) filamentous single-stranded DNA phages
D) lambda-based vectors
E) alpha-based vectors

Question 61

How can a protein be detected on a western blot?

Question 62

Explain the principle of riboswitches and switchboards, and provide examples of their application.

Question 63

The figure below depicts a "kill switch" or suicide module designed to eliminate an organism at a predetermined point, namely once the microorganism's function is accomplished. Explain how this kill switch can be regulated to prevent the untimely death of a microorganism harboring it.

Question 64

Discuss possible disadvantages of the yeast two-hybrid system.

Question 65

What is synthetic biology? Provide an example of its potential applications.

Question 66

Describe the use of a primary and secondary antibody in a western blot.

Question 67

What is the principle of the electrophoretic mobility shift assay (EMSA)? What is its main application? Mention a possible limitation.

Question 68

Briefly explain how real-time polymerase chain reaction (PCR) works.

Question 69

What is the conceptual basis of the primer extension technique?

Question 70

Describe the whole-genome DNA-binding analysis using ChIP-on-chip technology.

Question 71

How is the concept of "logic gates" used in synthetic biology?

Question 72

Observe the figure below and discuss how a single northern blot can reveal differences in size and abundance of the E. coli acid-resistance gene gadA with respect to the gadBC operon and the inducible expression of these genes at low pH.