Transformation Movement of DNA into bacteria Can use plasmid as vector Some bacteria are naturally competent and take up DNA from their surrounds Most bacteria are not, including E coli and we have to make it competent and get it to take up DNA from its environment Competency is a physiological state where bacteria can take up DNA from their environment Abuse bacteria with cold CaCl

When do the cutting and sticking of plasmid and foreign DNA there are several possible outcomes Successful sticking of the plasmid and foreign DNA Recircularization of plasmid without the foreign DNA Circulization of plasmid with other plasmids or several inserts to make huge circular molecule Many inserts sticking together to make long linear molecule All of these outcomes occur and could be taken up into bacteria Possibility 3 is less likely to happen as big plasmids are more difficult to take up and also less stable when they are taken up Possibility 4 can occur but linear DNA is usually broken down very quickly in a bacterial cell as it is recognised as non self

Selection A mechanism is required to select for the bacterial cells that have taken up the plasmid with one inserted foreign DNA piece Usually bacterial cells are used that are sensitive to a particular antibiotic The recombinant plasmid that is used usually carries antibiotic resistance and also a gene for Beta galactosidase. Beta galactosidase is an enzyme that acts on a substrate called X gal to yield a coloured product. The foreign DNA is inserted into a site in the middle of the beta galactosidase gene. This disrupts it and makes it inactive and unable to perform the colour reaction with X gal. Nutrient agar with ampicillin and X gal

Usually of bacterial origin (there are some eukaryotic plasmids). Plasmid vectors small circular dsDNA molecules capable of autonomous replication inside cells. Usually of bacterial origin (there are some eukaryotic plasmids). Designed Plasmids must have: origin of replication (Ori), Termination seqs Selective marker (usually antibiotic resistance gene) to select for bacteria containing plasmid. Polycloning site (unique restriction target sites useful for inserting donor fragments Many plasmids allow for blue/white selection for distinguishing between recombinant vs nonrecombinant plasmids polycloning site inserted inframe into beta-galactosidase gene (product of this gene converts colorless X-gal into blue product) Fragments inserted into this polycloning site disrupt function of b-galactosidase gene product, resulting in white colonies.                                              

plasmids used as vector when fragments of DNA to be inserted are smaller than 20 kb. Some plasmid vectors constructed such that cloned genes can be transcribed and translated. These are called expression vectors .

Types of Naturally occurring Plasmids All plasmids carry genes for their own replication – what other genes? No apparent function – cryptic plasmids for conjugation For antibiotic production Bacteriocin production Resistance genes Virulence genes Physiological functions: degradation of chemicals/herbicides, production of acetone, nodulation in symb N2 fixation

Resistance Plasmids R plasmids – resistance to antibiotics and other growth inhibitors Genes encode proteins that inactivate Ab or affect its uptake Can transfer resistance via conjugation Many drug resistance elements are on transposons Plasmid R100 carries resistance to sulfonamides, streptomycin, spectinomycin, chloramphenicol etc and can transfer itself to Klebsiella, Proteus, Salmonella and Shigella

Transfer of DNA between Cells In prokaryotes DNA transferred to recipient cell by 1 of 3 processes: Transformation: involves donor DNA free in environment Transduction: donor DNA transfer mediated by a virus Conjugation: involves cell to cell contact between donor and recipient i.e. acquisition directly from another bacterium