1. 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

3. 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

4. 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

6. 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.
                                             

7. 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 .

8. 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

9. 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

11. 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