QUIZ 9 Question 1 1 out of 1 points What is the purpose of having rrnBT4 in an expression vector? Selected Answer: Enhances the stability GOI transcript Answers: Enhances binding by transcriptional activators Enhances the stability GOI transcript Provides translation termination in 3 reading frames Allow high level transcription of the GOI Promotes strong binding of the ribosome to the translation initiation region Question 2 1 out of 1 points Having codons in the gene of interest that are not preferred in the expression host can lead to: Selected Answer: amino acid mis-incorporation Answers: full-length protein product Reduced mRNA degradation by ribonucleases Increased recombinant protein production Efficient translation amino acid mis-incorporation Enhanced mRNA stability Question 3 1 out of 1 points One source of the gene of interest for cloning is cDNA. What step is not part of cDNA synthesis? Selected Answer: Genomic DNA is isolated from the nucleus of eukaryotic cells Answers: ribonuclease is used in one of the steps to get rid of mRNA polyT primer is used to prime synthesis of cDNA dNTPs are added to the 3' end of primers Genomic DNA is isolated from the nucleus of eukaryotic cells DNA polymerase synthesizes the second complementary DNA strand Question 4 0 out of 1 points Why must reverse transcriptase be used to create a eukaryotic expression library? Selected Answer: Reverse transcriptase is able to add convenient cloning sites Answers: The resulting cDNAs do not contain non-protein coding introns Reverse transcriptase synthesizes double stranded RNA that is ligation-ready. Only reverse transcriptase can replicate eukaryotic DNA Reverse transcriptase ensures the gene is in the correct orientation in the expression vector Reverse transcriptase is able to add convenient cloning sites Question 5 1 out of 1 points What does not apply to restriction enzymes? Selected Answer: Restriction enzymes are proteins that restrict the activity of ligases Answers: Restriction enzymes are proteins that restrict the activity of ligases Some restriction enzymes generate single-stranded DNA overhangs Most restriction enzymes work as dimers Restriction enzymes break phosphodiester bonds between two nucleotides Restriction enzymes are a type of endonucleases Question 6 1 out of 1 points What is not associated with the colony blotting technique? Selected Answer: "Lysis of cells on the blot using the detergent, SDS, and the salt, calcium chloride" Answers: Denaturation of DNA that's on the blot Growing of bacteria containing the genomic libraries on agar with antibiotic for selection Transfer of transformed bacteria from an agar plate and onto a membrane "Lysis of cells on the blot using the detergent, SDS, and the salt, calcium chloride" Use of an X-ray film for autoradiography Use of a radio- or fluorescently labelled DNA probe to hybridize with the target gene Question 7 1 out of 1 points "As a new hire in a biotech company, you prepared the medium to be used for plating of E. coli after transformation. You added ampicillin to the medium, but got distracted, and forgot to add X-Gal. What of the following statements will apply? " Selected Answer: All colonies will be white Answers: No colonies will grow on the plates Only cells with gene X inserted in the cloning vector will grow All the colonies will be b-galactosidase positive All colonies will be white All the colonies on the plate will be ampicillin sensitive Only cells that don’t have gene X inserted in the cloning vector will grow Question 8 1 out of 1 points "After cleavage of the formyl-methionine from the N-terminus of a peptide, certain amino acids remaining on the N-terminus will make the protein susceptible to proteases. Which is not one of those amino acids?" Selected Answer: V - Valine Answers: Y- Tyrosine L-leucine W- Tryptophan V - Valine F-phenylalanine Question 9 1 out of 1 points Which type of ligation-independent cloning technique takes advantage of the recombination between lambda phage attB and attP sequences in the vectors and in the DNA fragment to be inserted? Selected Answer: Gateway cloning Answers: Restriction cloning Recombineering Exonuclease-mediated cloning Topo cloning Lysogenic cloning Gateway cloning Question 10 0 out of 1 points What will increase protein levels by increasing mRNA levels? Selected Answer: Secreting recombinant protein to periplasm where there are less proteases Answers: Having fusion partners at tne C-terminus of the protein Mutating sequences found in an operator region Removing proteolytic sites in the gene of interest by site-directed mutagenesis Mutating sequences in the GOI that blocks ribosome access to the start codon Secreting recombinant protein to periplasm where there are less proteases Question 11 1 out of 1 points "In the construction of recombinant DNA, what will increase the number of recombinant plasmids over non-recombinant plasmids?" Selected Answer: Increasing the insert:plasmid ratio Answers: Treating the fragment containing the gene of interest with alkaline phosphatase Cutting the plasmid with a blunt cutting restriction enzyme Using two different restriction enzymes with compatible overhangs Increasing the insert:plasmid ratio Phosphorylating the cloning vector Cutting the plasmid with a single restriction enzyme which generates sticky overhanging ends Question 12 1 out of 1 points What is true of alkaline phosphatase? Selected Answer: Answers: Alkaline phosphatase works on both strands of a cut DNA fragment Alkaline phosphatase removes the phosphate group from the 3’ ends of restrictionenzyme digested DNA strand Alkaline phosphatase transfers a phosphate group from a sensor protein to a transcriptional response regulator Alkaline phosphatase works on both strands of a cut DNA fragment Alkaline phosphatase promotes the formation of phosphodiester bonds between the restriction-enzyme digested plasmid ends "In restriction cloning, both cloning vector and insert are treated with alkaline phosphatase" QUIZ 10 Question 1 1 out of 1 points Which of these strain engineering approaches will not increase mRNA levels of the gene of interest? Selected Answer: Increasing expression of a gene for a repressor that binds to the GOI operator region Answers: Introducing T7 RNA polymerase for GOI driven by T7 promoter Increasing expression of a gene for a repressor that binds to the GOI operator region Deleting EcoKI Mutating rne1 Deleting recA Question 2 1 out of 1 points "When inoculating a bioreactor with starter culture, to avoid a long lag phase, the inoculum should be at what growth stage?" Selected Answer: Exponential phase Answers: Start of death phase Mid-stationary phase Exponential phase Middle of death phase Late stationary phase Also at lag phase Question 3 1 out of 1 points Which E. coli strain improves translation of heterologous genes by overexpressing genes for tRNAs that are rare in E. coli? Selected Answer: Rosetta Answers: DH5alpha Rosetta BL21 Star TRex BL21(DE3) Origami Question 4 1 out of 1 points Deletion of the genes for gor and _______ in the E. coli Origami strain enhances the formation of disulfide bonds in recombinant proteins expressed in the cytoplasm. Selected Answer: trxB Answers: groEL ompT arcA trxB fhuA2 Question 5 0 out of 1 points Proteins are very fragile biomolecules. During downstream product recovery and capture what is not a practice which will help stabilize proteins? Selected Answer: All the other choices will help stabilize proteins Answers: Add proteases All the other choices will not help stabilize proteins All the other choices will help stabilize proteins Add antibiotics to minimize bacterial contamination Work at low temperatures as much as possible Use appropriate buffers Question 6 1 out of 1 points Which step is not part of downstream bioprocess? Selected Answer: Optimizing pH of the growth media Answers: Optimizing pH of the growth media Chromatographic purification Filtration Dialysis or buffer exchange Vial filling and lyophilization Centrifugation Question 7 1 out of 1 points What is an advantage of fed-batch to continuous culture? Selected Answer: Mutations in the gene construct is less of a concern Answers: There’s better automation and process control in fed-batch Fed-batch yields a more uniform product quality "There’s more savings in time, labor, and energy with fed-batch" Fed-batch has higher volumetric productivity Mutations in the gene construct is less of a concern Question 8 0 out of 1 points "Which gene, when deleted, might decrease degradation of recombinant proteins?" Selected Answer: rne Answers: lonA rne fhuA2 groEL arcA Question 9 1 out of 1 points "Overexpressing chaperones, like groEL, can increase recombinant protein levels in E. coli by:" Selected Answer: Reducing levels of misfolded proteins Answers: Improving carbon utilization of E. coli Reducing levels of misfolded proteins Enabling cell cultures to reach higher cell densities Improving the stability of recombinant plasmids Increasing the DNA templates for transcription Prolonging the half-life of mRNA transcripts Question 10 1 out of 1 points "After centrifugation, aside from ethanol, what can be used to precipitate or concentrate the recombinant protein from the supernatant?" Selected Answer: Ammonium sulfate Answers: Sodium hydroxide Ethidium bromide Ammonium sulfate Thioredoxin Dithiothreitol Glutathione Question 11 1 out of 1 points When is the best growth phase to start inducing the expression of recombinant proteins? Selected Answer: Early stationary Answers: Early logarithmic phase Early stationary Before end of stationary phase Start of death phase Late decline phase Lag phase Question 12 0 out of 1 points The recA gene when knocked out may lead to increased recombinant protein production by: Selected Answer: Prolonging the half-life of mRNA transcripts Answers: Reducing mRNA degradation Prolonging the half-life of mRNA transcripts Reducing recombinant protein degradation Reducing levels of misfolded proteins Decreasing proteolysis of recombinant proteins Improving the stability of recombinant plasmids QUIZ 11 Question 1 1 out of 1 points What is not true about chymosin? Selected Answer: Chymosin is a lipase used to promote the curdling of milk Answers: Chymosin is an enzyme used in cheesemaking A recombinant chymosin was the first enzyme produced by recombinant DNA technology that was approved by the FDA for food use "Before recombinant biotechnology, chymosin had to be purified from calf stomach" Chymosin is a lipase used to promote the curdling of milk "Chymax is recombinant chymosin produced in fungus, Aspergillus" Question 2 1 out of 1 points "In the chromatographic separation used in the purification of recombinant human insulin peptides fused with b-galactosidase sequences, antibodies immobilized in the column beads were used to capture the fusion proteins. The type of chromatographic separation is:" Selected Answer: Affinity chromatography Answers: Ion exchange chromatography Mass chromatography Size exclusion chromatography Hydrophobic Interaction chromatography Affinity chromatography Question 3 1 out of 1 points What is not true about Pyrolase? Selected Answer: Pyrolase is an enzyme that retains activity at very acidic (pH 2) conditions Answers: Pyrolase has optimum enzymatic activity around 100oC "Native pyrolase is produced by the hyperthermophiic archeon, Pyrococcus furiosus" Pyrolase is an enzyme used in the oil industry to thin out the fracking solution Pyrolase is an enzyme that retains activity at very acidic (pH 2) conditions Pyrolase is a cellulase that breaks down polysaccharides Question 4 1 out of 1 points "___________________ is the largest product of industrial fermentation ton-wise, and is mainly used for ___________________." Selected Answer: Ethanol:fuel Answers: Petroleum: fuel Polyvinyl:plastics MSG:flavoring Ethanol:fuel Xanthan gum: Thickener Acrylamide:plastics Question 5 1 out of 1 points "An improved version of nucleoside phosphorylase which gave a higher yield of inosine or guanosine monophosphate, was produced by performing which type of mutagenesis technique?" Selected Answer: Error-prone PCR using Taq polymerase Answers: CRISPR-CAS9 Flippase-FRT UV mutagenesis Error-prone PCR using Taq polymerase Transposon mutagenesis Marker exchange using Flippase-FRT Question 6 0 out of 1 points "Which approach might increase formation of inclusion bodies in E. coli during recombinant protein expression, instead of decreasing them?" Selected Answer: Grow E. coli cultures at 25oC instead of 37oC Answers: Secrete recombinant protein to periplasm Employ a weaker promoter for the GOI "For lac promoter driven GOI, decrease lactose concentration" "For GOIs that are driven by CAP-regulated promoters, reduce glucose in the media " Overexpress certain chaperones Grow E. coli cultures at 25oC instead of 37oC Question 7 1 out of 1 points What is not true of inclusion bodies? Selected Answer: IBs consist mostly of highly active proteins Answers: IBs consist mostly of highly active proteins "For disulfide bond stabilized recombinant proteins, IBs can be lessened by secreting the protein to the periplasm" Inclusion bodies form due to the cell folding machinery being overwhelmed by protein overexpression Inclusion bodies form due to high numbers of exposed hydrophobic amino acids IB formation can be decreased by overexpressing chaperones Question 8 1 out of 1 points In ion-exchange chromatography: Selected Answer: Negatively charged proteins binds strongest to a positively charged stationary phase Answers: The largest proteins elute last from the column and the smallest ones come out first Hydrophobic proteins elute fastest from the column Negatively charged proteins elute slowest from similarly charged resins Negatively charged proteins binds strongest to a positively charged stationary phase The elution buffer gradient goes from high to low salt concentration QUIZ 12 Question 1 1 out of 1 points What does not apply to the regulation and process of T-DNA transfer by Agrobacterium tumefaciens to plant cells? Selected Answer: virD4 synthesizes the complementary strand of the T-DNA once inside the plant cell Answers: virB codes for the pili component that serves to attach A. tumefaciens to plant cells VirD2 is a relaxase that nicks the left and right border of the T-DNA (transfer) DNA. virD4 synthesizes the complementary strand of the T-DNA once inside the plant cell "Upon binding by chemicals that leak out of plant wounds, the sensor kinase, VirA, autophosphorylates in a conserved histidine residue" virE2 are single-strand-DNA binding proteins which protects the T-DNA from plant nucleases "The response regulator, VirG, gets phosphorylated by VirA and goes on to activate expression of other vir genes" Question 2 1 out of 1 points "To engineer -carotene production in Golden Rice, genes from the bacterium, Erwinia uredovora, and _____________________were transformed using Agrobacterium. " Selected Answer: daffodil or corn Answers: corn dandelion daffodil daffodil or corn squash carrots Question 3 1 out of 1 points What is not true about FrostBan or ice-nucleation active (INA) protein? Selected Answer: Answers: "In the presence of wild-type P. syringae, ice crystals begin forming on leaves at lower temperatures than in the absence of the bacterium" Inactivation of ina gene renders P. syringae less damaging on plants Spraying FrostBan extends the winter growing season of treated crops "In the presence of wild-type P. syringae, ice crystals begin forming on leaves at lower temperatures than in the absence of the bacterium" INA protein causes freezing injury to plant tissues Mutated ina gene was transformed and recombined into pathogenic P. syringae to make it avirulent Question 4 1 out of 1 points VectorVax protects turkeys from Newcastle disease virus. How was it made into a vaccine? Selected Answer: Answers: NDV gene was encapsulated in the coat protein of less-virulent fowlpox virus NDV was passed several times into non-host rabbits until an attenuated NDV was produced NDV was passed into media deficient in iron to produce a weakened NDV NDV was treated with formaldehyde to kill NDV NDV gene was encapsulated in the coat protein of less-virulent fowlpox virus NDV virulence genes were inactivated by marker exchange mutagenesis to weaken NDV Question 5 1 out of 1 points "The barnase gene from Bacillus amyloliquefaciens, is engineered in GM plants to: " Selected Answer: Make transgenic plants that have non-viable pollen Answers: Make transgenic plants that have non-viable pollen Make transgenic plants which are resistant to insect pests Make transgenic plants which are resistant to viruses Make transgenic plants which are resistant to nematodes Make transgenic plants which are resistant to herbicides Question 6 0 out of 1 points What is not true about FlavrSavr tomato? Selected Answer: FlavrSavr was the first recombinant food approved by the FDA Answers: FlavrSavr was the first recombinant food approved by the FDA FlavrSavr was genetically engineered to delay fruit ripening in tomato "In FlavRSavR, the target gene was inactivated using cre-loxP site-specific recombination " "FlavrSavr has decreased levels of polygalacturonase, a cell-wall degrading enzyme" FlavrSavr is edible Question 7 0 out of 1 points The plant resistance to viruses in more recently engineered transgenic plants is due to: Selected Answer: The coat protein interfering with the assembly of viable virus particles Answers: The transgene reducing the levels of viral mRNAs The coat protein interfering with the assembly of viable virus particles The coat protein causing a systemic immune response in transgenic plants The transgene interfering with the integration of the viral genome into the plant genome The coat protein interfering with the entry of the virus into the plant cells Question 8 1 out of 1 points Avermectin is produced by Streptomyces avermitilis and kills insects and worms by: Selected Answer: blocking transmission of electrical signals between nerve and muscle cells Answers: "killing cells lining the intestines thereby leading to insect starvation, then death" interferes with proper food digestion blocking transmission of electrical signals between nerve and muscle cells interfering with molting blocking the production of exoskeleton Question 9 0 out of 1 points What does not apply to the lysostaphin gene that has been engineered into cows: Selected Answer: leads to increased milk production Answers: kills Staphylococcus aureus codes for a peptide that has antimicrobial properties leads to increased milk production prevents mastitis is from a Bacillus strain Question 10 1 out of 1 points "Phy9X is an optimized version of the E. coli enzyme, phytase (AppA). Which of the statements below is incorrect about Phy9X?" Selected Answer: Answers: "Phy9X catalyzes the removal of nitrates from phytate, thus making nitrates available for absorption by livestock." "In Phy9X, there are 9 amino acids that have been changed from the native E. coli phytase " Phy9X in feeds increase retention of dietary metals by pigs "Phy9X catalyzes the removal of nitrates from phytate, thus making nitrates available for absorption by livestock." Site saturation mutagenesis was used to make amino acid changes in the wild-type appA. Phy9X remains active longer at lower gastric pH than the wild-type protein. Phy9X has a longer half-life at higher temperatures than the wild-type protein. Question 11 0 out of 1 points What is not true of inactivated vaccines? Selected Answer: Answers: Inactivated vaccines have lower risk of reversion to pathogenic state compared to attenuated vaccines Inactivated vaccines can be prepared by treating pathogens with chemicals like formaldehyde Inactivated vaccines are less immunogenic than attenuated vaccines Inactivated vaccines have lower risk of reversion to pathogenic state compared to attenuated vaccines Inactivated vaccines are designed to replicate slowly in animals Inactivated vaccines can be prepared by autoclaving pathogens Question 12 1 out of 1 points One of the very first crop engineered to be resistant to plant viruses was engineered to be resistant to PRSV. PRSV stands for: Selected Answer: Papaya Ringspot Virus Answers: Papaya Ringspot Virus Pineapple Roughshod Virus Potato Ringworm Virus Papaya Roughspot Virus Pineapple Ringspot Virus Question 13 1 out of 1 points What is not true of RNA-induced gene silencing? Selected Answer: RISC stands for RNA-Induced Splicing Complex Answers: The RISC loaded with short antisense ssRNA binds to target mRNAs. The slicer component of RISC cleaves the target mRNA. RNA silencing can result from blockage of translation of target mRNA. RISC stands for RNA-Induced Splicing Complex "Double stranded RNA is cut into short pieces by the RNase, DICER." Question 14 0 out of 1 points Which statement is true regarding the use of plant versus animal cell for producing biotherapeutic protein production? Selected Answer: Extraction and purification of proteins from animal cells are more complicated Answers: Animal cell cultures require less complex growth media "Using plants,there is little need for process optimization and monitoring" Animal cells require less expensive facilities and equipment to grow Cheaper to produce pharmaceutical proteins in mammalian cell cultures than in plants Extraction and purification of proteins from animal cells are more complicated Contamination of animal cell cultures by animal viruses is less of a concern
