Vectors

Question 1

What is a vector? What does they do?

Answer 1

The DNA into which the foreign DNA fragment is cloned

Act as the carrier
Increase the conc of the selected recombinant

Question 2

What are the types of vectors depending on their ability to survive inside the host? Describe

Answer 2

Stringent control - replicates with the host chromosome (coupled with chromosomal replication)

Relaxed control - replicates independent of the host. When chromo once – vector many. Has its own ori. Conc increase faster.

Question 3

What are the vector types depending on the host? What determines it?

Answer 3

Single host - only 1 ori

Multi host - shuttle vector - 2 ori

Ori region determines the host cell specificity

Question 4

Why only certain sized DNA is permitted?

Answer 4

Transient pors are for a short time. Only small mols can enter

Question 5

What are the propertis of cloning vectors? Describe

Answer 5

Ori

Polylinker

Selection genes

Question 6

What are the instances cloning vectors are used other than cloning?

Answer 6

Propagation of dna

Manipulation of dna

Delivery mode of dna

Question 7

What us a plasmid?

Answer 7

ds

Question 8

what are the classes of plasmids?

Answer 8

fertility-F-plasmids

resistance-R-plasmids

Col-plasmids

degradative plasmids

virulence plasmids

Question 9

Plasmid cloning vectors:

how the replicator is obtained? example?

Answer 9

from a natural molecule

Question 10

Plasmid cloning vectors:

dominant and recessive examples for selectable marker gene?

Answer 10

dom - Amp, Tet, Kan

rec - LeuB

Question 11

What are the 2 types of replicons. Describe.

Answer 11

conjugative transmissible - integrate into the chromosome

non-conjugative non-transmissible
eg: Col E1

Question 12

Disadvantages of using plasmid as the vector?

Answer 12

long time for recomb. selection

cant accommodate long fragments

relatively low copy no.

limited resc. sites - cloning potential limited

Question 13

Describe the steps in replica plating

Answer 13

pBR322 + Bam H1 >

add cut fragment(Sau3 A1) >

transform CaCl2 42C 2min - 20ng plasmid | 10^7 E.coli cells

plated out in Amp medium - 37C o/n

replicated onto Tet medium plate using velvet covered disc 37C o/n

compare 2 plates

Question 14

How pUC vector is made? Show the steps

Answer 14

Using pBR322

Tet gene removed
Ori of Col E1 is modified to be more efficient - mutagenesis
Lac Z’ gene introduced
Polylinker placed within lac z’

Question 15

How to screen recombinants of pUC ?

Answer 15

x-gal - blue/white selection

Question 16

In E.coli there is already a lac operon with lac Z in the chromosome. How is this distinguished from the lac z’ vector?

Answer 16

use mutant e.coli with contains chromosomal lac z which codes only for the omega fragment of beta-galactosidase

Question 17

What are the elements required in expression vectors?

Answer 17

Strong promoter

Rib binding site, terminator

Question 18

What type of vectors are used? Why is that?

Answer 18

Inducible
Can be toxic in hi conc.

Off by placing it dwnstream of a inducible promoter

Question 19

T7 phage properties & advantages?

Answer 19

Ss

Question 20

T7 promoter & T7 RNA pol. properties?

Answer 20

ss

Question 21

What is the vector used in expression by T7 RNA pol.? Its properties & advantages/disadvantages?

Answer 21

pET vector

strongest of promoters

Question 22

Describe the steps of joining target dna with pET vector, 2 component system?

Answer 22

ss

Question 23

Describe the process of expressing a gene with high frequency using pET vector?

Answer 23

BL21 DE3 ecoli contains T7 gene downstream of Plac

lac I gene express lac repressor which bind to Olac

IPTC remove lac repressor and induce transcription of T7 gene – T7 RNA polymerase

pET vector also has a lac I gene – lac repressor – but IPTG induces it

T7 rna pol binds to pET Plac

transcribe the target gene

Question 24

What is the vector that can use for further T7 regulation? What does it do?

Answer 24

pLysS vector

produce T7 lysozyme - bind and block T7

when IPTG is added lysozyme is degraded

Question 25

Name a repressible operon and list its special characteristics.

Answer 25

araBAD

-/+ regulation by the same protein

Question 26

Draw the structure of arabinose(araBAD) operon

Answer 26

ss

Question 27

Explain how the regulation of araBAD operon works when arabinose & glucose is not present?

Answer 27

araC synth. by rna pol – dimer – araBAD expression lower basal level

cAMP present – CAP-cAMP

araC binds to araO2 and araI1 forming a loop preventing rna pol binding

at high araC concs it binds to araO1 and block its own araC expression – *autoregulatory

Question 28

Explain how the regulation of araBAD operon works when arabinose is present & low glucose – active operon

Answer 28

arabinose binds to araC – weakens binding to araO2 – induce binding to araI2

RNA pol can bind to PBAD

low g - high cAMP - CAP-cAMP – bind to CAP site > this causes the loop to break > increase affinity of araC for araI2

araC-arabinose complex binds to araO1 and repress araC expression

RNA pol is activated > transcribe araBAD - breaks down arabinose

Question 29

How pBAD vector cloned genes are controlled?

Answer 29

tight repression when absent

strong activation when present

Question 30

What are suitable hosts fro pBAD?

Answer 30

TOP10

Question 31

What are the advantages & disadvantages of pBAD vector?

Answer 31

tightly controlled

strong inducibility

inexpensive induction

disadv-
not very high level of expression