Plasmids
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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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Plasmids

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Plasmids

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


Plasmids

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


Plasmids

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.


Plasmids

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


Plasmids

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


Plasmids

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.


Plasmids

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


Plasmids

  • 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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