Plasmids

• plasmid:

  • genetic element that exists outside the chromosome
  • replicates autonomously, independent from the host
  • circular
  • can be small and also large
  • dsDNA (double stranded)
• natural plasmid:

  • occur naturally in bacteria
  • e.g. E.coli
• artificial plasmid:

  • engineered usually from parts of natural plasmids
  • use/g take desired genes
  • e.g. pUC19
• ColE1 plasmid:

  • oriV = origin of replication
  • oriT = origin of conjugal transfer
  • rom = copy number control
  • mob = nuclease for mobilisation
  • colE1 = colicin E1 toxin
  • imm = immunity to colicin E1
• cloning plasmids:

  • artificial
  • vectors
  • different sides of the plasmid ring code for different phenotype genes
• plasmid evolution:

  • plasmids are dynamic
  • 1 = invasion where there is exchange of DNA from host to plasmid which will incorporate itself into the host
  • 2 = genome evolution where DNA incorporated from bacteriophage due to conjugation
  • 3 = host range
  • 4 = peristence and antibiotic resistance
  • 5 = transition
• characteristics of plasmids:

  • circular dsDNA
  • multiple only inside a host cell
  • size varies
  • carry range of number of genes
• copy number:

  • small plasmids have high copy number = replicate faster than cell division and random segregation into daughter cells
  • large plasmids have lower copy number = replication coordinated with chromosome and is tightly controlled otherwise plasmid is lost
• plasmid partitioning:

  • several mechanisms
  • actin filaments
  • tubulin filaments
• plasmid addiction systems:

  • kills cells that lose the plasmid
  • hok/sok system in E.coli
  • hok protein will kill cells, if plasmid lost = rapid sok = degrade as less stable without stable hok
• conjugative plasmid:

  • F plasmid
  • sexual transfer via a sez pilus
• mobilisable plasmids:

  • cannot initiate conjugation
  • can be transferred by conjugation in the presence of a conjugative plasmid
• non mobilisable plasmids:

  • only transferred by natural transformation or transduction
• horizontal transfer:

  • transduction = use of bacteriophages to pick up plasmid from host cell and incorporate into genome
  • transformation = natural/common incorporation of free DNA into their chromosome
  • conjugation = sex pilus to transfer plamid from one cell to another compatible cell
• F plasmids:

  • fertility
  • tra genes confer ability to express sex pili and conjugate
• R plasmids:

  • resistance
  • provided resistance to toxins and antibiotics
• col plasmids:

  • encode bacteriocins for killing other bacteria
• degradative plasmids:

  • allow metabolism of unusual substances e.g. pollutants
• virulence plasmids:

  • convert bacteria into pathogens
• transferability:

  • where certain plasmids can move themselves from one bacterial cell to another
  • usually F plasmids
• mobilisability:

  • small plasmids will not be able to transfer from one host to another
• incompatible plasmids:

  • they are plasmids that belong to the same family
  • they cannot be sound in the same host as they will compete with each other
• virulence plasmids:

  • tumour inducing
• yeast plasmids:

  • 2 micrometer circle
  • found in nucleus of most strains
  • 6.3kb circular
  • stable
  • no phenotype
  • useful for genetic engineering
  • high copy number
• plasmid vectors:

  • origin of replication = replication in host
  • selectable marker = antibiotic resistance (maintenance of high copy number)
  • multiple cloning site = unique restriction enzymes
  • expression vector = promoter whch can be induced
  • fusion tag = affinity purification
• shuttle vector:

  • 2 origins of replication
  • propagation of 2 hosts
  • cloning in E.coli, expression in yeast