Presentation on theme: "Plasmids Plasmid - an extrachromosomal circular DNA molecule that autonomously replicates (has an Ori ) inside the bacterial cell; cloning limit: 100 to."— Presentation transcript:
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
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
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.
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
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
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.
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
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