Vectors 1:Plasmids Flashcards
How are clones typically generated?
Placing a DNA fragment of interest into vector DNA which can then replicate in a host cell
What are plasmids?
Extrachromosomal self-replicating DNA molecules
Circular dsDNA molecules
Exist in multiple copies in bacterial cells
Function in nature is to enhance adaptation of bacteria to the environment by transferring genes horizontally without the need for conjugation
Responsible for outbreaks of antibiotic-resistant pathogenic bacteria in hospitals
What are the 2 main features of plasmids?
A replication origin, enabling the plasmid to be replicated independently of the bacterial genome
A gene that confers a selective advantage on the organism and selects for the plasmid
How are plasmids replicated?
Replication places a metabolic load on the organism and thus plasmids tend to be spontaneously lost when no advantage is gained by their presence
Are passed on to daughter cells when fission of bacterial cells occurs
What does the nature of origin define?
The replication group of the plasmid
Only one plasmid of a given replication group can be stably maintained in a bacterial cell
How are plasmids purified?
After cell lysis, centrifuge and will have different sedimentation to genomic DNA in a density gradient
How are plasmids reintroduced into bacterial cells?
Bacterial cells are made leaky by using a treatment with permeabilising agents (Ca2+)
These cells are then called competent
Competent cells are then mixed with DNA and subjected to mild heat shock
What is electroporation?
Electric shock treatment to increase the uptake of DNA
What is blue/white selection?
E.coli cells are engineered to synthesise an incomplete, inactive enzyme ω-peptide
Cells containing the original vector plasmid also synthesise a small peptide fragment from β-galactosidase - α-peptide
α-peptide and ω-peptide bind together to give an active β-galactosidase
Cells with the original plasmid will produce a blue colony when treated with X-Gal
In plasmids with an insert, the gene is interrupted and the α-peptide is mutated and the colonies are white
What type of DNA is taken up by plasmids?
Circular DNA
Where does the insert go in the plasmid?
Inserted and doesn’t replace a part
How efficient is the transformation?
Good (10^7μg^-1) and gets worse with larger insert sizes thus is better for smaller inserts
