6. Bacterial Genetics III Engineering Genes and Cells

  1. molecular cloning
    isolating a specific gene from the genome and making many copies, usually by propagating it in a plasmid
  2. why do molecular cloning
    • used to understand the function of the gene:
    • move gene into a different organism, different mutant background
    • change its regulation
    • specifically mutate that gene
  3. basic steps in molecular cloning
    • cut foreign DNA (can be chromosome if target gene is unknown or PCR product if target gene is known) and vector with same restriction enzyme to create complimentary sticky ends
    • add DNA ligase to form recombinant molecules
    • introduce recombinant vector into a host using transformation or conjugation
    • select transformants that are resistant to vector antibiotic marker
  4. what are restriction enzymes and why do bacteria have them?
    • protect from foreign DNA (often phage genomes)
    • restriction endonuclease cuts specific, palindromic DNA sequence, they are dimers with two active sites
    • modification systems - methylase protects host DNA by methylating a base within the same sequence, blocking the restriction enzyme
  5. vector
    • plasmid used to carry your gene of interest
    • have multiple cloning sites or polylinkers containing several unique restriction enzyme sites for insertion of a DNA fragment
    • designed to help you to help you distinguish empty vectors from those containing inserted DNA
  6. alpha-complementation (blue/white screening)
    allows selection of transformants containing a vector and screening for vectors that contain an insert
  7. background for understanding alpha-complementation
    • host cells makes nonfunctional B-gal (white) empty vector - encodes missing a-peptide that restores B-gal activity (blue)
    • inserting a fragment into the multiple cloning site of a vector interrupts the coding sequence of the alpha-peptide
    • transformants with vectors containing foreign, inserted DNA will remain B-gal-, while those contain empty vectors will become B-gal+
    • detect beta-galactosidase activity using an indicator called X-gal, which creates a blue color when it is cleaved by B-gal
  8. requirements for plasmids - they are designed to work with specific strains
    • host DNA replication machinery must recognize plasmid origin of replication
    • host strain must be sensitive to antibiotic you are using for selection
    • for blue/white screening, host strain must synthesize only the truncated form of B-gal
  9. How do you identify the affected gene in a mutant?*
    complementation of mutant phenotype using genes carried on plasmids - used for identification in random mutagenesis

    • If recessive mutation:
    • Make genomic DNA library from wild-type parent

    Transform mutant with the library

    select for presence of plasmid (antibiotic marker) and growth at non-permissive restriction (ex high temperature)

    colonies that grow should contain a vector holding the wild-type gene

    re-isolate plasmid and sequence the DNA
  10. How do you identify the affected in a mutant if the mutation is a dominant mutation?
    • transform with genomic library and select for transformants on antibiotic medium
    • replica plate onto identical plates and grow one at permissive temperature and other at non-permissive temperature
    • screen for transformants that grow at permissive temperature sequence insert that was cloned into vector from each transformant
    • compare the vector sequence obtained with the sequence from the wild-type chromosome to identify the mutant
  11. When do you use random mutangenesis and what can you use?
    • when i don't know at all what genes are involved in my process of interest
    • have no sequence information about my organism

    tools - chemical mutagens, UV radiation, transposons
  12. When do you use targeted mutagenesis and what can you use?
    • when I can predict what genes will be involved in my process of interest
    • you must have sequence information about the genes you are targeting or a whole genome sequence

    tools - targeted gene disruption to create knockout mutations
  13. How do you create knockout mutations by targeted gene disruption?
    • clone gene into a plasmid
    • cut gene with restriction enzymes
    • replace internal region with antibiotic resistance gene
    • leave 500-1000 bp at each end so homologous recominbation can occur between sequences and identical ones at chromosomal locus
  14. What can you do to ensure recombination with vector in targeted gene disruption?
    • linearize plasmid before transformation
    • used a vector that cannot replicate in the host
  15. forward genetics vs. reverse genetics
    • forward:
    • make random mutations (easier)
    • select or screen for mutants with desired phenotype

    • reverse:
    • make directed mutations in genes of choice (harder)
    • test each mutant in collection for desired phenotype
  16. transposon mutagenesis
    • have to mutagenize at random
    • select mutants that received a transposon hop
    • select or screen for mutants with your desired phenotype

    • does not gurantee a mutation in a specific gene
    • can insert into any DNA sequence
    • do not use homologous recombination
Card Set
6. Bacterial Genetics III Engineering Genes and Cells
general microbiology midterm 1