Lecture 221)Describe the main features of the F plasmid a) Fertility plasmid; contains genes about how to conjugateb) Can transfer itself or the donor chromosome into a recipient cell2)Describe what Hfr strains are a) High frequency recombination; strains where the F plasmid is integrated into the cells genome 3)Explain how Hfr strains can transfer chromosomal DNA to recipient cells a) The plasmid replicates with the chromosome, the Tra region can still cause DNA transfer4)Compare and contrast conjugation from F+ and Hfr strains a) F+i) DNA being transferred: F plasmidii) Recipient cell Becomes F+ iii) Steps(1) Donor cell contacts recipient through sex pilus(2) Pilus retracts(3) Cells make contact, F plasmid is nicked at oriT(4) One strand of F is transferred to recipient, F replicates in donor cell(5) Synthesis of second strand in recipient cell by rolling circle (6) Completion of DNA transfer and synthesis, cells separate b) Hfri) DNA being transferred: Segment of F plasmid + segment of chromosomeii) Recipient cell stays F- + chromosomal genes(1) The F plasmid is nicked in one strand(2) F is transferred followed by chromosomal DNASynthesis of second strand in recipient and donor cells 5)Describe what oriT is a) Origin of transfer; site on the genome where the plasmid breaks to travel through the sex pilus6)Describe how replication of the F plasmid during conjugation is different from chromosomal DNA duplication during cell division a) Enzymes and proteins are used to assist with DNA replication, two cells do not have a sex pilus interaction. And DNA replication is done be elongation and initiation not rolling circle replication 7) Explain what restriction enzymes area) They cut DNA at specific sequences 8) Compare and contrast sticky ends and blunt ends a) Sticky ends is where one strand sticks out more than the other and blunt ends are double stranded everywhere and even.9) Name two applications of PCRa) Research- cloning, mutagenesis, phylogenetic studiesb) Diagnostic: detection and identification of microbial infections 10) Explain what molecular cloning is a) Inserting a DNA fragment into a molecule that can be replicated and manipulated11) Describe the three main features of plasmid cloning vectorsa) Usually contain multiple cloning sites (MCS) for cloning DNA fragmentsb) Carry one or more antibiotic resistance genesc) Have an origin of replication independent from the chromosomed) Exist as multiple copies per cell12) Describe what expression vectors area) Contain genetic elements that optimize the expression of cloned genes13) Describe what shuttle vectors are, and what feature allows them to exist in multiple host species a) Replicate different organisms, such as bacteria and eukaryotic cellsb) Has multiple cloning sites and genetic constructs can be made in e coli and transferred to the organisms of interest.14) Explain what merodiploidy and complementation are a) Merodiploidi) A cell that has two copies of a gene inside of itb) Complementation i) If two copies of a gene are present and one is damaged the other can take itsplace 15) List two applications of site-directed mutagenesis a) Used to study enzyme function. Can be used to study the importance of individual amino acids in enzyme activity, in specificity of the enzyme for its substrate etc.b) Used to give new properties to enzymes 16) List the three steps of site-directed mutagenesis a) Step 1: PCR with the template plasmid and the mutagenic primersb) Step 2: Diegestion of the PCR mixture with the restriction enzyme Dpnl. Dpnl cuts only at methylated restriction sites. Only the parental plasmid is methylatedc) Step 3: Transform PCR product in E. coli to recover mutant plasmid 17) Explain why digestion with the restriction enzyme DpnI increases the efficiency of site-directed mutagenesis a) Diegestion with restriction enzyme Dpnl cuts only methylated restriction sites. Only the parental plasmid is methylated18) Compare and contrast transposons and insertion sequences a) Transposonsi) Transposons plus other genes flanked by two insertion sequencesb) Insertion sequencesi) Transposase gene flanked by two inverted repeatsii) Found on chromosomes and plasmids 19) Explain what genes, besides transposase, are often found in transposons a) Transposons- some genes flanked by two insertion sites b) Transposable virus20) Compare and contrast conservative and replicative transpositions a) Conservative i) The transposon is excised from the chromosome and reinserts at a new locationb) Replicativei) A duplicate copy is made; this copy integrates at a new location: the original remains 21) Explain one application of transposons in genetic research a) Very useful for bacterial genetic analysis- create mutations by gene
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