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188 Cards in this Set
- Front
- Back
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What is the central dogma?
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DNA --> RNA (transcription) --> protein (translation)
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For each of the following, state whether it is required for transcription or translation: shine delgarno sequence, ribosome, RNA polymerase, Rho/hairpin formation, tRNA, start codons, promoter.
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translation, translation, transcription, transcription, translation, translation, transcription
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Name all types of RNA.
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rRNA, tRNA, mRNA, ncRNA, sRNA
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What are the 4 levels of regulation?
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transcriptional, post-transcriptional, translational, post-translational
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Which technique allows for the comparison of transcription profiles under different conditions?
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DNA microarrays
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T/F. Bacterial genome size is highly flexible.
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T
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Is a bacteria with a genome of about 10^7 base pairs likely to be free-living or host-dependent?
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free-living
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T/F. There is a good correlation between chromosome size and number of genes.
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T
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Approx. how many more genes are there than Mb of chromosome?
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1000x more
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Which types of bacteria are often richer in GC?
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free-living
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Which types of bacteria likely have higher mutation rates?
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host-dependent
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T/F. Bacteria with large genomes and high GC content live in fairly stable environments therefore do not need to mutate as often to adapt.
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F. They live in variable/complex environments
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What is an operon?
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many genes under the same promoter sequence
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T/F. Genes under the same operon share transcription and translation.
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F. Share transcription but translation is independent (have their own RBS)
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How can looking you tell if two genes are under the same operon just by looking between them?
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Of there are <35 nucleotides between them there is likely not a promoter between them therefore they'd be under the same operon
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T/F. The promoter is located after the start codon.
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F
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T/F. Promoters are AT rich
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T
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T/F. The terminator sequence lies before the stop codon.
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F
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Which codons are stop codons?
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TAG, TAA, TGA
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The RBS is rich in which nucleotide(s)?
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A and G
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What is a Rho-independent terminator sequence followed by?
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T rich region
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Why are internal RBSs avoided?
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they hinder transcription
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Compare plasmids and chromosomes.
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plasmids are often smaller in size, have non-essential genes and can be passed to other species horizontally
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T/F. Megaplasmids are roughly the size of the smallest bacterial chromosome.
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F. megaplasmids are about 3x bigger
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What is essential for chromosome compaction?
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nucleoid-associated proteins; supercoiling
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Which enzyme relaxes DNA and which introduces supercoils?
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topoisomerase; gyrase
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Which type of supercoiling does bacterial DNA undergo?
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negative
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T/F. Condensins make the daughter DNA molecules harder to separate.
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F. easier to separate
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What are SMCs and Muk proteins?
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condensins
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T/F. H-NS, HU, Fis and IHF are all histones.
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F. They are histone-like proteins
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Which nucleoid-associated protein is the most like histones?
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HU
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What does H-NS stand for?
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heat-stable nucleoid-structuring protein
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Where does H-NS bind and what does it do?
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to AT-rich sequences and links/bridges DNA
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What are the N and C domains of H-NS involved in respectively?
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N: protein-ptotein interactions, C: DNA-binding
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Compare DNA and RNA polymerase.
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DNA polymerase used in replication, requires 3'OH group (primer), requires a template, synthesizes in 5'-3' direction. RNA pol is involved in transcription and does not require a 3'OH group
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Where does DNA replication start?
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at the AT rich oriC
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Which enzymes synthesizes the new DNA during DNA replication?
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DNA pol III
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Which enzyme removes primers and fills in gaps during DNA replication?
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DNA pol I
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What must happen in order for there to be a large enough gap for SSBP to come in during strand separation? How big is the gap?
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DnaA binds oriC; ~20 nt
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How many nucleotides must a gap be in order for helicase to come in?
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~45 nucleotides
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Helicase causes unwinding of about ____ nt which allows ___ to be loaded on the strand.
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65; primase
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What were the 2 possibilities for DNA pol III localization? How was this tested? Which one is accurate?
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variable (moves along the DNA() or constant (DNA moves but pol stays put); PolC-GFP fusion; constant
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What is PolC?
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catalytic subunit of DNA pol III
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Is PolC-GFP a transcriptional or translational fusion protein?
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translational (transcribed on same transcript and translated as one unit)
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Where are the start codon(s), stop codon(s) and RBS(s) on the PolC-GFP fusion protein located?
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`start codon before PolC, stop codon after GFP, RBS before start codon
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How can you check that your fusion protein is functional?
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put it in a cell with a TSM in the protein of interest, both should be expressed at permissive temperature but only plasmid copy at non-permissive temperature
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How can you adjust your fusion if it is not functional?
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attach GFP to a different subunit, play with the linker, attach GFP to the N-terminus instead of the C-terminus
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Where would your fusion protein be located if GFP was fused to DnaA?
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oriC
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Where would your fusion protein be located if GFP was fused to DnaB?
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slightly ahead of DNA polymerase
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Where would your fusion protein be located if GFP was fused to primase?
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one on leading strand, many on lagging strand, all before DNA pol
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Where would your fusion protein be located if GFP was fused to DNA pol I?
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behind DNA polymerase III
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What is the secondary role of DNA polymerase in nature?
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chromosome segregation
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What is needed for a succesful PCR reaction?
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dNTPs and DNA pol III
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Why are DNA pol I and ligase not required for PCR?
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DNA primers are used instead of RNA primers so they don't need to remove them and ends of the lagging strand do not need to be joined
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What are the approximate denaturation, annealing and extension temperatures for PCR?
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95; 60;72
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Which PCR temperature depends on the length and composition of primes?
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annealing temperature
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Which PCR temperature depends on how far apart your primers are?
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extension time
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Why might you want to amplify a coding sequence and its upstream regulatory elements but remove the stop codon?
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if you're amplifying a fusion product
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Why might you want to amplify a coding sequence with its downstream regulatory elements but not its promoter or RBS?
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if you're putting it into a plasmid that already has them
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T/F. Upstream primer must be directly before the promoter if entire gene and all reg elements being amplified.
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F. works better if its a little more upstream since TFs often act upstream of the promoter
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Where would your primer 2 be located if you're trying to amplify a sequence without its stop codon?
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just before the stop codon
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Where would your 1st primer be if you want to amplify a coding seuqnce without its upstream elements?
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after the promoter, at the start codon
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How long should primers be?
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18-24 bps
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What is the general rule for annealing temperature?
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Tm = 2(A+T) + 4(G+C)
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What is ideal at the 3' end of a primer?
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2-3 GCs (GC clamp)
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Which primer end is flexibe?
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5'
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What is the ideal melting temperature range for primers?
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55-70 deg C
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How can you determine if your PCR reaction worked?
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run on a gel
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What does it mean if you get 2 bands when running your PCR product on a gel? What is the best way to fix this?
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primer annealing elsewhere also; raise the annealing temperature
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What are the 3 main components of plasmid cloning vectors?
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origin of replication, selection marker, multiple cloning site
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Describe endonucleases.
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recognize specific DNA seq (4-8bp), usually short inverted repeat that is palindromic, cleaves both strands of DNA
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State whether BamHI, Sau3AI and SmaI leave blunt or sticky ends.
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sticky, sticky, blunt
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What are the pros and cons of blunt-end cloning?
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it is straightforward but there is no control over cloning direction
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At which end are restriction enzymes introduced?
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5' end
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T/F. Restriction enzyme sequences are included in melting temperature calculations.
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F. It is not annealing initially
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How many nucleotides are added before restriction endonuclease sites?
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any 4-6 nucleotides
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Which type of cloning includes restriction endonuclease sites in primers?
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directional
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Is the product of directional cloning sticky or blunt ended?
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blunt
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What must happen to the directional cloning product before it is cloned into a plasmid?
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must get digested by restriction enzymes (same with the plasmid)
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Where on the plasmid does the cloning product get integrated?
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multiple cloning site
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What is different about primers used when performing site-directed mutagenesis?
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both contain a mutation in the middle
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Which property of an amino acid is more important for protein function?
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site-directed mutagenesis (substitute other amino acids and see if they can restore function)
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Why might a DNA polymerase with a higher mutation rate be useful?
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if the organism has many mutations, normal polymerases won't really work since mismatched bases can kink out the DNA
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What are the 3 possible scenarios when DNA polymerase makes an error?
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1) incorrect base replaced with correct one, extension continues 2) holoenzyme dissociates and reassociates to try extension again 3) holoenzyme does not fix mismatch (mutation)
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What is the role of DnaQ?
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exonuclease and subunit of DNA polymerase that corrects 90-99% of DNA polymerase errors
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An antimutator strain has ___ DnaQ activity and ___ mutation rate
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increased; decreased
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How can you test mutation rates?
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focus on mutations in one important gene with an obvious phenotype (ex.antibiotic resistance gene)
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What is the role of lacZ?
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encodes beta-galactosidase which cleaves lactose into glucose and galactose
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What is X-gal?
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lactose analogue, colonies turn blue when it gets cleaved
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What does IPTG do?
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turns on lacZ expression
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Would a mutator strain have more or less blue colonies in an x-gal screen than an antimutator screen?
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less blue colonies
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Are mutation rate estimations often over or under estimates and why?
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underestimates due to silent mutations
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Which mutant isolation method is useful for identifying a wide variety of mutants?
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screen
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Which mutant isolation method is best at isolating rare mutants?
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selection
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Why is DNA gyrase a good antibiotic target?
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not found in eukaryotes
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How many proteins make up the DNA gyrase complex?
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4
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What is the role of DNA gyrase? Is it essential for DNA replicaiton?
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relieves tension in DNA caused by helicase; yes
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Why antibiotic targets GyrA?
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ciprofloxacin
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Which antibiotic targets GyrB?
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novobiocin
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Is resistance to ciprofloxacin more or less common that resistance to novobiocin? Why?
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ciprofloxacin resistance is more commin because there are only 2 chances for mutations in GyrB but many more in GyrA
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Would wild type gyrA cells grow on a plate with ciprofloxacin?
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yes
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Why might mutator strain grow less well than the wildtype?
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still getting mutations in the rest of the cell
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Why would the anti-mutator strain grow less well than the wild type?
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replication takes longer
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How can spontaneous mutations occur?
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DNA pol errors, UV, free radicals
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What is an auxotrophic mutant?
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has a mutation in a metabolic pathway
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Would you screen or select for tryptophan auxotrophs?
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screen
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T/F. Tryptophan production requries many genes, all of which must be functional.
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T
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Describe the mutations causing temperature sensitive mutants.
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modest amino acid substitution
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Would you screen or select for TSM?
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screen
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Would you screen or select for ciprofloxacin resistance?
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select
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T/F. Some amino acids are more flexible with codons than others
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T
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Where do nonsense mutations have the greatest effect? The least effect?
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beginning of a gene; regulatory region
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Where do missense mutations have the greatest effect? The smallest effect?
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about the same throughout the gene; regulatory region (not translated)
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T/F. It is not possible to have frameshift mutations in regulatory regions.
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T
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When is a frameshift mutation the most deleterious?
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at the beginning of a gene
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Which experiment determined if mutations are spontaneous or specific responses?
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Lura and Delbruck
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What is TonB?
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receptor on E.coli to which phage T1 attaches
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What is special about TonB mutants?
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they are resistant to T1 phage
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Would you screen or select for TonB mutants?
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select
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What would your # of resistant bacteria column look like when plated on plain plates or ones spread with T1 phage? Why?
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about equal; flask is mixed
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What would you expect if you plated an aliquot from each culture on a different plate spread with phage T1 if mutations are random? Why?
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different numbers of resistant bacteria; because it depends which cell in the lineage has the mutation
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How can you calculate mean number of mutants?
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mean = m/n
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How can you calculate the number of mutations per tube?
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m = -lnPo
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What is Po?
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number of tubes containing no mutants / number of tubes
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What is the formula for mutation rate?
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a = m/N
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What does N represent when calculating mutation rates?
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number of cell divisions/cells
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What are the pros and cons of spontaneous mutations?
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pro: variety, con: could take a whike
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Give a pro and a con of induced mutations.
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pro: control over mutagen (concentration, chemical reactivity, time) con: heavy mutagenesis could kill all cells (increased likelihood of hitting on essential genes)
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How could you study radiation resistance?
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heavy mutagenesis
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Would you screen or select for mutations in an essential gene?
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screen
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When/why would you perform an enrichment?
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rare mutations, mutant doesn't grow under selective conditions but doesn't die either
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What does ampicillin do?
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targets growing peptidoglycan therefore only kills actively growing and dividing cells
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T/F. Mutant cells cannot grow and divide under enrichment conditions.
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F
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T/F. Enrichments can be used to determine mutation rates.
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F
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What is the end result of an enrichment?
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higher proportion of mutant cells to wt cells
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T/F. Enrichment cannot be performed for selections.
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F. Selections are the ultimate enrichments
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Which of the following can you perform and enrichment for: mutation in essential gene, in biosynthetic pathway, conferring antibiotic resistance, in gene required for catabolic pathway
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all of the above
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How can you figure out where a mutation is after screening/selecting/enriching for it?
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complementation
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Describe the complementation process.
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w/t chromosome cut partially and at common sequence, ligated to plasmid cut with same sticky ends, transform into mutant cells, select for cells with plasmid (w/t phenotype restored), PCR amp plasmid chromosome fragment, sequence it
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Which types of mutations are often recessive?
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loss of function
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T/F. It is difficult to complement tryptophan mutants.
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T
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What is a revertant?
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chromosome with mutation mutates back to w/t
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T/F. Supressors have the same sequence as the w/t.
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F
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Which type of mutations are easiest to revert?
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point mutations (deletions are pretty hard)
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What are the 2 classes of suppressors?
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intragenic and intergenic
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Describe an intragenic suppressor.
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revertant can change to a less deleterious amino acid (still not id to wt though), inactive promoter can suppress active site mutation, same codon but diff aa, restored reading frame
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What are intergenic suppressors?
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can alter amount or activity of mutant protein, can alter a different pathway (asp substituted for glu), affect mutant pathway (increase aa uptake if production disrupted)
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What does vanomycin target? How can resistance be acquired?
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peptidoglycan; VanHAX enzymes remodel the cell wall
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What is the role of VanS?
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senses vanomycin
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What is the role of VanR?
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initiates membrane cascade stimulating vanHAX transcription
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What happens when there is a muation in vanS preventing it form phosphorylating VanR? How can vanomycin resistance be restored?
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strain is vanomycin sensitive; mutation in vanR causing VanR to be constantly active
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SUPPRESSOR ISOLATION??
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L10
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T/F. Plasmids always contain circular DNA.
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F
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T/F. Plasmid replication is dependent on chromosome replication.
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F. they are independent
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Plasmid replication is controlled by ____-encoded genes but requires __ replicaiton machinery.
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plasmid; host's
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What determines host range and plasmid copy number?
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origin of replication
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Plasmids that initiate replication ___ of host are said to have broad host range.
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independently
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How is plasmid copy number regulated?
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1) antisense RNAs 2) antisense RNAs + protein 3) binding of replication proteins to iterons
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UNDERSTAND REPA COPA COPB TAP
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L11
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SLIDE 12
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L11????
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Describe type I plasmid partioning.
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ParB binds to parS, ParA associates with the ParB of one plasmid and the ParB of another and polymerizes between plasmids to force them apart
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Describe type II plasmid partioning.
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ParR binds to parC, ParM associates with ParR on each plasmid and forms a mitotic-spindle-like filament to separate the plasmids
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Describe plasmid addiction.
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toxin kills cell once plasmid is lost since antitoxin on plasmid is lost also
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What are hok and sok?
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hok encodes small toxin, sok encodes anantisense RNA
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Are toxins or antitoxins more stable?
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toxins
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Why would a cell die if a plasmid containing the toxin and the antitoxin is lost?
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hok mRNA is still present (no sok to degrade it) therefore it still gets translated
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Why might two plasmids be incompatible?
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have identical oriVs and regulatory machinery, share the same par functions (can't differentiate between them)
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What happens when 2 plasmids share same replication/partioning?
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one plasmid gets lost (cell cured of that plasmid)
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What do you need to create a cloning vector? What is optional?
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origin of replicaiton, selection marker, multiple cloning site; origin of transfer
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Where on pUC18 is the multiple cloning site?
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within lacZalpha
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What does lacI do?
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represses lac operon in absence of lactose
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What must be expressed in order to get beta-galactosidase activity?
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both the alpha (plasmid) and beta (on chromosome) domains
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Would blue colonies indicate that an insert is or is not present?
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is not
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What does a cloning vector have that a suicide vector does not?
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an origin of replication
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What are suicide plasmids useful for?
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gene disruptions and duplications
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What is sacB and when is it often used?
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gene that confers death in presence of sucrose; in suicide plasmids, used to select for the 2nd crossover
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Would cells with single crossovers live or die in the presence of sucrose when sacB is present on the suicide plasmid?
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die
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What is conjugation?
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process by which plasmids transfer themselves from one cell to another
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T/F. Cell contact is required for the transfer of genetic information.
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T
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T/F. DNA transfer is unidirectional and conservative.
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T
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What are the 2 types of conjugative plasmids?
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self-transmissable and mobilizable
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Which enzymes get transferred to the recipient during conjugation?
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relaxase and primase
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What are Mpf genes?
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mating pair formation genes encode the pilus
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Where does relaxase attach and what does it do?
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oriT, nicls DNA and reseals it once inside the recipient
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Which type of replication results in transfer of a single DNA strand to the recipient?
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rolling circle replication
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What is an Hfr strain?
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bacterial strain with a self-transmissible plasmid integrated into its chromosome
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Does the recipient cell obtain the F plasmid upon mating with an Hfr strain?
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no, the chromosome containing the plasmid is rarely fully transferred
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How long does Hfr chromosome transfer take?
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~100 mins
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