Plasmids

Question 1

Why are supercoils formed?

Answer 1

To relieve strain from underwound or overwound DNA

Question 2

Topoisomerases are

Answer 2

enzymes that change topology of DNA

Question 3

Type 1 topoisomerases

Answer 3

cleave 1 strand of a double helix -> unwinds supercoils

Question 4

Type 2 topoisomerases

Answer 4

Cleave 2 strands of a double helix -> introduce supercoils

Question 5

Mechanism of unwinding supercoil

Answer 5

1 strand cleaved, enzyme holds onto both ends and passes in tact strand through break for religation

Question 6

How negative supercoils are introduced with DNA gyrase (TPII)

Answer 6

1. GyerB grabs 1 section
2. GyerA introduces double-stranded break + becomes covalently attached holding 2 ends apart
3. GyerA ( ATPase) uses ATP to pass in tact double-stranded section through break
4. Gyer B rejoins cleaved DNA + opens to release strand

Question 7

A low copy number means

Answer 7

Only a small # of plasmids allowed in daughter cell -> each daughter gets 1

Question 8

How to ensure stringency in low copy number?

Answer 8

Attach plasmid to cell membrane so only 1 plasmid per daughter cell -> attachment generates signal

Question 9

Why is incompatibility in plasmids needed for cloning?

Answer 9

2 plasmids with similar replication/ seg. mechanisms can’t co-exist in bacterium

Question 10

Outline rolling circle replication:

Answer 10

1. Replisome contains tyrosine which holds phosphate
2. Replisome makes a nick b cloning DSD + holds onto PD backbone
3. End of nick (3’OH) is getting synthesised by DNA polymerase (helicase separates strands) while replisome at 5’ end drags along
4. Nick between both strands: DSD and SS -> sealed by replicase
5. ligase -> 2 species
6. SS -> DSD -> since there’s no free 3’OH DNA primase is used at SSO then DNAPIII will add + extend to form CCC DNA

Question 11

What is the mechanism which mediates replication in controlling copy #?

Answer 11

1. Helicase disrupts H-bond to create a fork
2. RNA II binds to prevent DNA strands from binding -> RNA-DNA hybrid
3. RNAseH -> a polymerase I degrades RNAII regions
4. 3’OH of RNAII primes DNA synthesis
5. Replication

Question 12

What is the mechanism which prevents replication in controlling copy #?

Answer 12

1. RNA1 is produced and is complimentary to RNAII
2. ROP acts as a dimer and dimerises RNA I and II
3. Duplex formation drags RNAII away from OriT
4. Fork collapses and complimentary DNA sequence re-binds
5. No RNAII-DNA hybrid so no rep

Question 13

PBR322 characteristics

Answer 13

• 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

Question 14

Why is it important that 1089 bp removed from PBR322 to make PBR327?

Answer 14

No residual conjugative properties so can be biocontained

Question 15

How is plasmid amplification measured?

Answer 15

3H/14C ratio when 3H added post-wash and plasmid grown in 14C -> extent of DNA replication can be measured

Question 16

How can we improve amplification in plasmids with low copy number?

Answer 16

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.

Question 17

We can separate plasmids by amount coiled how?

Answer 17

Buoyant density equilibrium centrifugation -> the more coiled, the more dense so lower band

Question 18

Ampicillin acts on

Answer 18

cell wall synthesis

Question 19

Tetracycline acts on

Answer 19

30s ribosome

Question 20

Zeocin acts on

Answer 20

DNA strand degradation

Question 21

Other antibiotics can

Answer 21

bind to 70s ribosome

Question 22

Lambda phage K is

Answer 22

a Lambda phage C which survived in E.coli K by methylation, only for 1 cycle

Question 23

Type 1 restriction endonuclease

Answer 23

Multi-subunit -> recognise, cleave and methylate, needs ATP, Mg2_ and s-adenyl methionone

Question 24

Type 2 restriction endonuclease

Answer 24

Methylates separate 3’OH and 5’ phosphates

Question 25

Type 3 restriction endonuclease

Answer 25

Multi-subunit, sites have to be in inverse orientation

Question 26

Type 4 restriction endonucleases

Answer 26

Only modified DNA