MOLECULAR BIOLOGY MIDTERM EXAM NEWEST 2024/2025 ACTUAL EXAM QUESTIONS AND CORRECT DETAILED ANSWERS

MOLECULAR BIOLOGY MIDTERM EXAM NEWEST 2024/2025 ACTUAL EXAM QUESTIONS AND CORRECT DETAILED ANSWERS What are the key differences between strains used for DNA (plasmid) work vs protein expression work? - AnswerCloning vectors are used to carry foreign DNA segments into a host cell while expression vectors contain suitable expression signals with maximum gene expression What are three genetic markers that would indicate a strain that is used for DNA replication? Why? - AnswerGenetic Markers Dam: Blocks adenine methylation at GATC sequences which makes DNA susceptible to cleavage by some restriction sites endA1: Activity of nonspecific endonuclease I is abolished which improves yield and quality of isolated plasmid DNA mutS: Deficient in mismatch repair which stabilizes DNA heteroduplexes during site-directed mutagenesis What are three genetic markers that would indicate a strain that is used for protein expression? Why? - AnswerompT: Strains harboring this mutation are deficient in outer membrane protease VII, Reduces degradation of expressed recombinant protein DE3: λ lysogen that encodes T7 RNA polymerase - induces expression in T7 drive expression systems. Presence of a polymerase to act as a landing pad for the promoter to attach and begin transcription lacIq: Mutation in the lac repressor gene for constitutive expression. Inhibits transcription from the lac promoter, which can be overcome by IPTG addition . Allows controlled gene expression from promoters that carry the lac operator Bacterial culture conditions can impact plasmid preparation yield and quality. What are the issues that can decrease your DNA miniprep (any method)? - Answeri) low culture volume: Low volume = less cells = less plasmid/DNA ii) Poor culture media: Choose the right broth/richer media - poor culture = less cell density = less plasmid dna yield overall iii) Too short of an incubation time: Too short of incubation time = less cell density = less cells to grow = less yield Describe the three buffers used in bacterial lysis used in plasmid DNA purification (Qiagen spin or anion exchange columns). - Answeri) P1 Buffer = Resuspension buffer to resuspend the cells