Plasmids Flashcards
Why are supercoils formed?
To relieve strain from underwound or overwound DNA
Topoisomerases are
enzymes that change topology of DNA
Type 1 topoisomerases
cleave 1 strand of a double helix -> unwinds supercoils
Type 2 topoisomerases
Cleave 2 strands of a double helix -> introduce supercoils
Mechanism of unwinding supercoil
1 strand cleaved, enzyme holds onto both ends and passes in tact strand through break for religation
How negative supercoils are introduced with DNA gyrase (TPII)
- GyerB grabs 1 section
- GyerA introduces double-stranded break + becomes covalently attached holding 2 ends apart
- GyerA ( ATPase) uses ATP to pass in tact double-stranded section through break
- Gyer B rejoins cleaved DNA + opens to release strand
A low copy number means
Only a small # of plasmids allowed in daughter cell -> each daughter gets 1
How to ensure stringency in low copy number?
Attach plasmid to cell membrane so only 1 plasmid per daughter cell -> attachment generates signal
Why is incompatibility in plasmids needed for cloning?
2 plasmids with similar replication/ seg. mechanisms can’t co-exist in bacterium
Outline rolling circle replication:
- Replisome contains tyrosine which holds phosphate
- Replisome makes a nick b cloning DSD + holds onto PD backbone
- End of nick (3’OH) is getting synthesised by DNA polymerase (helicase separates strands) while replisome at 5’ end drags along
- Nick between both strands: DSD and SS -> sealed by replicase
- ligase -> 2 species
- SS -> DSD -> since there’s no free 3’OH DNA primase is used at SSO then DNAPIII will add + extend to form CCC DNA
What is the mechanism which mediates replication in controlling copy #?
- Helicase disrupts H-bond to create a fork
- RNA II binds to prevent DNA strands from binding -> RNA-DNA hybrid
- RNAseH -> a polymerase I degrades RNAII regions
- 3’OH of RNAII primes DNA synthesis
- Replication
What is the mechanism which prevents replication in controlling copy #?
- RNA1 is produced and is complimentary to RNAII
- ROP acts as a dimer and dimerises RNA I and II
- Duplex formation drags RNAII away from OriT
- Fork collapses and complimentary DNA sequence re-binds
- No RNAII-DNA hybrid so no rep
PBR322 characteristics
- artificial plasmid vector 1977
- similar to pUC19 but has tet gene
- derrived from PMBI
- combines 3 plasmids with ampR and tetR
- reproduction rate increased, conjugal transfer size reduced by removal of 2 PstI sites from PBR313
Why is it important that 1089 bp removed from PBR322 to make PBR327?
No residual conjugative properties so can be biocontained
How is plasmid amplification measured?
3H/14C ratio when 3H added post-wash and plasmid grown in 14C -> extent of DNA replication can be measured
How can we improve amplification in plasmids with low copy number?
Use chloramphenicol for low copy # plasmids e.g. CoIEI and pMB1 -> protein synthesis is inhibited in host chromosome. But plasmid replication is independent of newly synthesised proteins so continues for several hours until 2000-3000 copies accumulated. Rifampin stops plasmid rep.
We can separate plasmids by amount coiled how?
Buoyant density equilibrium centrifugation -> the more coiled, the more dense so lower band
Ampicillin acts on
cell wall synthesis
Tetracycline acts on
30s ribosome
Zeocin acts on
DNA strand degradation
Other antibiotics can
bind to 70s ribosome
Lambda phage K is
a Lambda phage C which survived in E.coli K by methylation, only for 1 cycle
Type 1 restriction endonuclease
Multi-subunit -> recognise, cleave and methylate, needs ATP, Mg2_ and s-adenyl methionone
Type 2 restriction endonuclease
Methylates separate 3’OH and 5’ phosphates
Type 3 restriction endonuclease
Multi-subunit, sites have to be in inverse orientation
Type 4 restriction endonucleases
Only modified DNA
