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Plasmids Plasmid - an extrachromosomal circular DNA molecule that autonomously replicates (has an Ori ) inside the bacterial cell; cloning limit: 100 to 10,000 base pairs or 0.1-10 kilobases (kb) 10 -100 copies per cell called high copy number 1- 4 copies, low copy number

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slide1

Plasmids

Plasmid - an extrachromosomal circular DNA molecule that autonomously replicates (has an Ori ) inside the bacterial cell; cloning limit: 100 to 10,000 base pairs or 0.1-10 kilobases (kb)

10-100 copies per cell called high copy number

1-4 copies, low copy number

Many exist naturally in

bacteria

Many recombinant forms

have been designed for

use in cloning

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Different kinds of plasmids that exist in nature

F plasmids (fertility)

  • Has genes for conjugation
  • Carries Tra genes for transfer and formation of sex pili

R plasmids

  • code for enzymes that result in inactivated antibiotics
  • can be many resistances on one plasmid
  • conjugative and permiscuous (spreads readily)

Sym plasmids

  • Rhizobia nodulate legumes
  • Fix nitrogen
  • Genes for nodulation and fixation on the sym plasmid

Col plasmids

  • Bacterial proteins that destroy closely related proteins

Metabolic plasmids

  • carry genes to degrade specific substances like toluene, pesticides

Virulence plasmids

  • Code for specific toxins and capsular proteins
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Recombinant plasmids
  • These have been designed to carry foreign DNA inserted into them into a cell.
  • They are a type of Cloning vector e.g.

1. pBR322

  • derived from 3 others
  • pSC101
  • pSF2124
  • pMB1
  • familiy of similar vectors
  • over 20 unique restriction sites
  • 12 of sites are in Amp R and TetR genes and their promoters
  • cloning into these sites makes selection of recombinants easier as it results in insertional inactivation of resistance genes
  • normal copy number is 15/cell
  • pBR324 and pBR 325 are plasmids derived from 322 but have insertional inactivation of different selectable markers.

2. pUC family of vectors

  • has a Lac Z gene that continues to produce beta galactosidase unless a foreign gene is inserted.
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Copy numbers
  • Generally want high copy numbers, exception is where high level of expression of protein has a lethal affect on host, then want low copy number.
  • pBR322 derivatives generally low copy number
  • Allows ‘lethal protein’ to be expressed below lethal concentration
    • Can increase copy number by
    • cultivating bacteria with plasmid under conditions such that protein synthesis is arrested e.g. use chloramphenicaol
    • some plasmids have a temperature sensitive mutation that leads to uncontrolled replication at high temps
    • ROP gene is involved in replication control if you remove that replication goes nuts

What are the ideal features of a cloning vector such as a plasmid?

  • replicates in host cell
  • unique restriction endonuclease cloning sites
  • at least one selection system
  • ds DNA
  • low molecular weight so room for big insert and not energetically costly to cell
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Steps in cloning a piece of DNA
  • obtain fragment
  • obtain plasmid
  • construct plasmid with insert
  • transform host cell with recombinant plasmid
  • screen for successful cells with recombinant plasmid with inserted foreign DNA
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Making a Recombinant plasmid

1. Sticky ends

2. poly tailing technique

  • allows any 2 DNA molecules to be joined by adding poly A to the 3’ ends of one piece and poly T to the 3 ‘ends of the other piece

3. Blunt end ligation

  • relies on ability of T4 ligase to join blunt ended molecules
  • advantage is no additional material introduced
  • not v. efficient
  • difficult to control which blunt ends are ligated

4. Chemicallly synthesized linkers can be made. Disadvantage is still have to blunt end to stick them on, advantage is can insert a Restriction site and can recover insert easily.

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Transformation
  • This is getting the DNA into the bacterial cell
  • Some bacteria are naturally competent and this is how DNA can move around in nature.

e.g. Streptococcus pneumonia (gm +ve)

    • cells secrete competence factor(CF) in exponential phase
    • this binds and stimulates the synthesis of 8-12 new proteins
    • one is autolysin which exposes DNA binding protein and nuclease on cell surface
    • DNA binds in a ds form , any DNA can bind
    • Nuclease hydrolyses one stand
    • Other strand associates with proteins and crosses the cytoplasmic membrane
    • May integrate into genome, a transformant is a cell with an altered genetic make up as a consequence of taking up external foreign DNA
    • If not integrated or not circular DNA will be degraded
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e.g. Haemophilis influenzae (gm –ve)
    • no competence factors
    • ds DNA enters cell as ds form
    • only DNA from close relatives can bind
    • DNA must contain specific 11bp sequence for binding
    • 600 copies in H influenza
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