Chapter 3: Cloning: Vectors & Transformation

Vector

nucleic acid molecule used to carry and replicate sequences of interest in a host organism

  • Replicate Independently of chromosomal DNA: Allow high copy # inside cell and ability to replicate
  • Unique cloning sites: Short segment of DNA with RE sites, allows researcher to ligate DNA specifically
  • Selectable markers: To detect the transformation
  • Easily recovered from host cell: Allows isolation of recombinant from host cell for reuse or remove

Plasmid Vectors

Circular, supercoiled DNA molecules existing as extrachromosomal elements

  • Small, circular 3-5kb: More room for inserts, more unique RE sites, not attacked by hose exonuclease
  • Origin of replication: Where replication begins, different types of plasmids with same ORI cannot co-exist as they compete for machinery and involved in copy number control
  • Multiple cloning site: A number of unique adjacent RE sites, allows multiple fragments in one region
  • Copy number control: Trait associated with each type of plasmid , number of plasmids per cell ORI
  • High copy number: To produce protein we want a lot of
  • Low copy number: To produce less, potentially toxic substance at high
  • Known Sequence: Aids DNA sequencing and predict RE sites and restriction maps
  • Selectable marker Detect transformations, cells that have plasmid will grow and screening too extensive
  • Insertional Activation: Screening technique allowing for detection of recombinant bacteria
  • Lac z is a partial galactosidase gene, the other lac Z coding region is supplied by host genome (mutant with deletion of part of lac z)
  • Alpha complementation: Both parts come together and create functional enzyme and turn blue in presence of xGAL substrate, if the insert is cloned into the multicloning site it interrupts and forces cells to be white
  • Uncut / Self Ligation = blue, no vector = nothing, small insert = could be blue small impact

Transformation

Uptake of naked DNA (free of protein) by a bacterial host

Competent

Cells capable of transformation, minimum efficiency of 107 transformants / ug to be effective

CaCl2 treatment followed by Heat Shock

  • Grow cells to log phase, collect by centrifugation wash and resuspend in ice-cold CaCl2, which will affect the cell wall, add DNA which binds to the surface, heat shock stimulates uptake and then LB media added
  • Positive charge of calcium neutralizes negative charge of plasmid and cell wall, therefore dissipating electrostatic repulsion and weakening the cell wall
  • Sudden increase in temperature (42oC for 30 seconds) crease a pressure difference between the outside and inside of cells, induces the formation of pores through which supercoiled plasmid DNA enters

Electroporation

  • Grow cells to log phase, centrifuge collect and wash, resuspend at high concentrations in 10% glycerol & freeze in small aliquots, mix frozen cells with DNA in cuvette, zap with high voltage briefly, add LB
  • Sterile water removes electrolytes, pulse of voltage causes pores to open allowing DNA to enter
  • Take longer to prepare but are faster to use and have higher transformation efficiency

Few competent cells take up recombinant plasmid, selectable markers such as ampicillin resistance used to detect

Vectors Based on Bacteriophage Lambda

Bacteriophage can take up inserts 5-20kb and cosmids can take up 35-45kb

  • Lambda genome is packaged into capsid as linear molecule (50000 bp) with 12 cohesive ends (Gs and Cs)
  • Lambda is linear in capsid and then goes through membrane and circularizes in the cell

Temperate virus

  • Lysogenic: Genetically silent, dormant, monitors metabolism of cell
  • Lytic: Infects, multiplies, then kills by lysis – detected by plaques on solid LB agar

Packaging of viral DN

  • Recognizes cos sites on concatemers and cuts DNA creating cohesive end and fills headful into capsid head
  • In-vitro packaging: Mix ligated DNA with lambda heads (packaging enzyme will recognize cos sites in DNA) to stuff heads and add tails
  • DNA by transfection
  • Temperature Sensitive: Raise temperature enough to infect then raise so it can’t go through life cycle and gets blocked at stage it was missing gene product and will accumulate heads or tails

Cosmids

  • Plasmid with cos site, invitro packaging, 32-45 kb inserts circularizes to form plasmid after infection

Artificial Chromosome Vectors

  • BACs YACs: new vectors developed to accept larger inserts upto 350 kb (entire genomes)


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