Deck 12: Bio-techniques and Synthetic Biology

Question 1

When using a luminescent reaction as part of the detection system in a western blot, released photons may be revealed by:

A) UV illumination
B) phosphoimaging
C) colorimetry
D) spectrophotometry
E) none of the above

Answer 1

B

Question 2

What end of the reporter DNA probe is bound to the reporter dye in real-time PCR?

A) 5'
B) 3'
C) amino-terminal
D) carboxy-terminal
E) none of the above

Answer 2

A

Question 3

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, etc.
E) none of the above

Answer 3

B

Question 4

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) none of the above

Question 5

The electrophoretic mobility shift assay (EMSA) is utilized to monitor molecular interactions between:

A) two proteins
B) RNA and protein
C) DNA and protein
D) DNA and RNA
E) none of the above

Question 6

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

A) transcriptional
B) translational
C) both transcriptional and translational
D) posttranslational
E) none of the above

Question 7

Acid-resistant Escherichia coli cells extrude H⁺ through the combined action of two proteins: a decarboxylase enzyme and the transporter:

A) H⁺ channel
B) [H⁺]-ATPase
C) GABA/glutamate antiporter
D) all of the above
E) none of the above

Question 8

The first historical record of biotechnology was:

A) the discovery of penicillin by Alexander Fleming
B) the use of bacteria in producing hormones such as insulin
C) the use of genetic engineering in production of vaccines by plants
D) the use of microorganisms in the manufacture of bread, alcohol, and dairy products
E) the discovery of DNA's double-helix structure and the genetic code

Question 9

Microbes that grow at neutral pH but can survive at pH 2 for short periods are called:

A) acid-fast
B) neutralophiles
C) acid-resistant
D) extremophiles
E) none of the above

Question 10

Which of the following is correct regarding gene fusions?

A) They are also called transcriptional fusions.
B) Only the target gene has its own promoter and ribosome-binding site.
C) They consist of an artificial single-promoter operon that produces two proteins.
D) Both target and reporter genes have their own promoter sequences.
E) Both target and reporter genes have ribosome-binding sites.

Question 11

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

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

Question 12

A significant advantage in real-time PCR is that:

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) all of the above
E) none of the above

Question 13

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

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

Question 14

The colorless molecule o-nitrophenyl galactoside (ONPG) can be used as an artificial substrate in assays of $\beta$ -galactosidase in E. coli extracts. Which of the following statements is NOT true with respect to the use of ONPG?

A) Hydrolysis of ONPG by $\beta$ -galactosidase produces yellow o-nitrophenol.
B) o-nitrophenol can be measured with a spectrophotometer.
C) Hydrolysis of ONPG by $\beta$ -galactosidase also releases colorless galactose.
D) The o-nitrophenol moiety is blue and more suitable for use in solid media.
E) None of the above.

Question 15

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 16

What kind of mutation is produced by transposable elements?

A) inversion
B) deletion
C) insertion
D) substitution
E) none of the above

Question 17

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- $\beta$ -gal fusion protein?

A) Yes. Hydrolysis of X-Gal by $\beta$ -galactosidase produces a blue product detectable by eye.
B) Yes. Hydrolysis of ONPG by $\beta$ -galactosidase in liquid medium produces an orange compound measurable by spectrophotometry.
C) Yes. It depends on how the construct is made with respect to the position of the RBS.
D) No. Whereas YFP fluorescence can be carefully observed and measured by time-lapse microscopy, $\beta$ -gal activity is measured as an average of the population as a whole.
E) None of the above.

Question 18

Infection of potato crops by the fungus Phytophthora infestans is a constant menace to farmers. A biotechnological approach to prevent the recurrence of such an infestation is:

A) cloning of a wild potato gene that conveys resistance to P. infestans into commercial potato breeds
B) frequently spraying potato plants with fungicide throughout the growing season
C) watering plants with a trickling mechanism to avoid excessive dampness
D) all of the above
E) none of the above

Question 19

The genes for proteins such as $\beta$ -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 $\beta$ -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 20

His₆-fusion-tagged proteins can be isolated by affinity chromatography because the histidine tag tightly binds to:

A) copper
B) magnesium
C) iron
D) calcium
E) nickel

Question 21

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 22

DNA fragments with specific sequences are detected by a technique known as:

A) northern blot
B) Southern blot
C) western blot
D) isoelectrofocusing
E) agarose gel electrophoresis

Question 23

Why must double-stranded DNA probes be denatured when used in northern blot experiments?

A) to ensure that they are properly radioactively labeled or tagged with a dye
B) to ensure that the DNA coding strand hybridizes to RNA on the membrane
C) to ensure that both DNA strands bind the single-stranded RNA on the membrane
D) all of the above
E) none of the above

Question 24

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 $\beta$ -mercaptoethanol

Question 25

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) all of the above
E) none of the above

Question 26

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) 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) separation of mRNA from other RNA species
E) none of the above

Question 27

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

A) their isoelectric point
B) their quaternary structure
C) their molecular weight
D) their amino acid sequence
E) their secondary structure composition

Question 28

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

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

Question 29

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) All of the above.
E) None of the above.

Question 30

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) none of the above

Question 31

Among the applications of the yeast two-hybrid method, one can find which of the following?

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) all of the above
E) none of the above

Question 32

Gene fusions of a protein of interest and GFP, or its derivatives, can be used in:

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) all of the above

Question 33

Potential vaccine antigens made by transgenic plants include:

A) hepatitis B surface antigen
B) E. coli heat-labile enterotoxin B subunit
C) rabies virus
D) all of the above
E) none of the above

Question 34

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) none of the above

Question 35

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 $\beta$ -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 $\beta$ -galactosidase catalyzed the hydrolysis of X-Gal.
E) Blue colonies are an indication that GAL4 activated transcription of GAL1-lacZ.

Question 36

A protein of interest can be specifically detected on western blots; this technique requires:

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) all of the above
E) none of the above

Question 37

The most common crops expressing insecticidal peptides are:

A) wheat and rice
B) tomato and potato
C) cotton and corn
D) all of the above
E) none of the above

Question 38

What hybridization technique can be used to confirm if two genes are components of an operon by estimating transcript size?

A) northern blot
B) Southern blot
C) formaldehyde cross-linking
D) agarose gel electrophoresis
E) isoelectric focusing

Question 39

How does the enzyme 5'-monophosphate-dependent terminator exonuclease (TEX) distinguish between bona fide start sites in primary transcripts and those resulting from internal RNA cleavage?

A) TEX hydrolyzes processed transcripts from their 5'-ribonucleotide monophosphates.
B) TEX hydrolyzes primary transcripts from their 5'-ribonucleotide monophosphates.
C) TEX hydrolyzes RNA starting from a primary transcript's 5'-7-methylguanosine cap.
D) TEX hydrolyzes processed transcripts from their 5'-ribonucleotide triphosphate 5'-ends.
E) TEX hydrolyzes primary transcript from their 5'-ribonucleotide triphosphate 5'-ends.

Question 40

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

A) to establish the position of the binding protein relative to the transcription start site
B) to provide molecular weight markers
C) to check that the gel running conditions are correct
D) to check for nonspecific DNA degradation
E) none of the above

Question 41

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 42

The following is a potential application of bacterial oscillator switches:

A) the study of controlled interactions between two feedback genetic loops
B) the design of bacterial genetic circuits to detect toxins
C) the design of bacterial clocks
D) all of the above
E) none of the above

Question 43

What is the role of kill switches in genetically engineered microorganisms?

A) They serve as a fail-safe mechanism to timely kill the organisms after they have fulfilled their purpose.
B) They lead to the suicide of the genetically modified organism to prevent their reproduction.
C) They potentially allow the release of engineered microorganisms outside the laboratory without the risk of biological contamination or invasion.
D) All of the above.
E) None of the above.

Question 44

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

Question 45

Among the uses for phage display are:

A) the identification of high-affinity peptides that bind to attachment proteins of some eukaryotic viruses and effectively block viral infection
B) antibody fragments cloned from natural sources
C) collections of mutant enzymes to be tested for activity
D) all of the above
E) none of the above

Question 46

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

Question 47

Describe the difference between autoradiography and phosphoimaging.

Question 48

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

Question 49

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

Question 50

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) none of the above

Question 51

Once a gene encoding a putative transcriptional regulatory protein is identified by sequence annotation, what needs to be done to demonstrate this activity?

Question 52

What is the biochemical mechanism of acid resistance in E. coli O157:H7?

Question 53

The use of BioBricks is advantageous, except that:

A) it encourages collaboration from laboratories around the world
B) it helps in producing new genetic logic circuits and functional systems
C) it stimulates "outside the box" thinking
D) anyone can do it and there may be unintended consequences
E) none of the above

Question 54

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) migration of negatively charged DNA in an electrophoresis experiment depends on molecular weight
C) energy-dependent transporters in the nuclear envelope
D) all of the above
E) none of the above

Question 55

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

Question 56

How can a single northern blot 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?

Question 57

Which of the following best describes a "buffer gate" in synthetic biology?

A) A single repressor molecule (R₁) prevents the production of an output molecule (Pr₁).
B) Alternate input molecules (I₁ or I₂) drive the production of alternative proteins (Pr_n).
C) A single input inducer molecule (I₁) leads to the production of a single product (Pr₁).
D) All of the above.
E) None of the above.

Question 58

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 59

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

A) it demonstrates that genetic manipulation of recalcitrant organisms is feasible
B) it opens the possibility to redesign prokaryotic genomic systems
C) it enables the engineering of new species
D) all of the above
E) none of the above

Question 60

What are crRNA and taRNA, and what is their role in riboswitches?

A) newly described small RNAs, part of the mRNA processing multiprotein RISC
B) previously unknown ribosomal RNA molecules that, when paired, release the RBS for translation
C) small RNA molecules that can control the translation of an mRNA of interest
D) all of the above
E) none of the above

Question 61

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

Question 62

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

Question 63

How can a protein be detected on a western blot?

Question 64

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

Question 65

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

Question 66

What is the conceptual basis of the primer extension technique?

Question 67

Discuss possible disadvantages of the yeast two-hybrid system.

Question 68

What is the basis for the use of the Bt toxin as an insecticide? What are some of the beneficial aspects of using Bt?

Question 69

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

Question 70

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