PLASMIDS Flashcards
How are plasmids visualised under an electron microscope?
They are coated in heavy metals and revealed using platninum shadowing
- cytochrome c binds to plansmid
What is supercoiled DNA?
How is DNA negatively supercoiled ?
Overwound or underwound DNA using isomerases
- puts a stress on the double helix and creates a deficit of turns
How does agarose gel electrophoresis work?
can be treated with topoisomerases to relax the DNA
- usually DNA is negatively charged, the supercoiled form of the DNA will travel faster than the relaxed form
RIGHT HANDED UNDERTWIST
Negatively supercoiled
LEFT HANDED OVERTWIST
Positively supercoiled
How does sedimentation analysis work?
Spin DNA in centrifuge and fill with a gradient of sucrose, the most compact DNA molecules will sediment faster
Open circular/relaxed dna will be second fastest. Dna that is broken/linear will sediment the most slowly
How does equilibrium density ultracentrifugation work?
fill centrifuge tube with solution of caesium chloride with the same density as dna. Then spin the tube for a long time, creating a gradient of caesium chloride. Density that is same as DNA is somewhere in the middle.
How can ethidium bromide be used in equilibrium density ultracentrifugation? Useful for?
Can add other chemicals to interact with DNA. For example, ethidium bromide can be added to intercalate between the bases of the dna and makes them untwist, and twist in the other direction.
Separating supercoiled DNA from non supercoiled
Describe the non-essential properties that are often coded for by plasmids
- drug resistance
- resistance to heavy metals
- pathogenicity
- nitrogen fixation
- antibiotic synthesis
- catabolism of aromatic hydrocarbons
- tumorgenicity in Agrobacterium
What does ampicillin resistance look like ?
Breaks the beta-lactamase ring via beta lactamase
What does chloramphenicol resistance look like?
uses transacetylase to acetylate CAM
What does tetracycline resistance look like?
decreased ability to concentrate tetracycline
Streptomycin resistance?
OH on streptomycin removed by ATP
Plasmid has 3 regions
1 for antibiotic resistance
1 as an origin of replication
1 for transfer functions
How does incompatibility arise?
from two plasmids having the same replication system
