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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

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Plasmids

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  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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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