Vectors II

Question 1

What are fusion proteins?

Answer 1

fused genes for 2 proteins

Question 2

What are the uses of fusion proteins? Describe

Answer 2

Identification

• antibody binding
• fluorescent

In quantification
-surrogate proteins

In purifications
-tag proteins bind to a particular substance

Question 3

List some examples fro

Answer 3

hexahistidine - high affinity to Ni
chitin
Arg-tag
S-tag
MBP
CBP
Amylose

Question 4

What are the 3 types of plant vectors?

Answer 4

based on natural occuring plasmids from Agrobacterium - Ti, Ri

direct gene transfer - various plamid

plant viruses - caulimo, gemini

Question 5

What happens in an A. tumefacians infection?

Answer 5

Ti-plasmid > T dna integrated into plant dna > tumors >. synthesize opines

Question 6

Show how Ti-Plasmid is used in plant cloning

Answer 6

ss

Question 7

Why phage vectors are better over plasmids? drawbacks?

Answer 7

plasmids have to transport. cant do big pieces but phages inject and propagate. infection is efficient than transformation

more complex
depend on bacteria

Question 8

Virus structure types?

Answer 8

head with tail - lambda

filamentous - M13

Question 9

Viral life cycle types?

Answer 9

lytic

lysogenic

Question 10

main steps of pattern of viral infection?

Answer 10

infection

replication

phage formation

Question 11

lambda phage properties? role of cos sites?

Answer 11

package

rolling circle replication

Question 12

Show how dna is packaged inside lambda phage

Answer 12

cos- concatamer

Question 13

Show how dna is replicated in lambda phage

Answer 13

dna injected

concatamer formation

head proteins + endonuclease A expressed

cuts dna @ cos

package into capsids

Question 14

What is the drawback of lambda phage as a vector? How is it minimized?

Answer 14

capsid – physical constraint on amount of DNA

by remove unnecessary parts and make designer vectors

Question 15

Draw & give exampls for insert. & subs. vectors

Answer 15

Lgt10
Charon 16A

Embl3
Embl4

Question 16

how a foreign gene is cloned into lambda phage vector?

Answer 16

t4 dna ligase

Question 17

how to do in-vitro packaging of a lambda vector? explain in steps

Answer 17

2 methods?

Question 18

READ MORE IN-VITRO PACKAGING!

Answer 18

READ!

Question 19

M13 phage characteristics?

Answer 19

ss

Question 20

What are the advantages of M13?

Answer 20

ds repli. form

ss during the infection

Question 21

Visualize the genome of M13 phage

Answer 21

ss

Question 22

Explain the life cycle of M13

Answer 22

ss

Question 23

Show how vectors are designed from M13 phage

Answer 23

m13mp1
m13mp2
m13mp7
m13mp8
m13mp9

Question 24

How to use M13 in cloning? explain with steps

Answer 24

ds RF M13 cloning
insert fragment into MCS
transfect into host cell
replication
ss DNA assembled
recombinant phage
>polethylene glycol
precipitated phage
isolated by adding phenol
ss recombinant dna

Question 25

What is the selection medium for M13?

Answer 25

X-gal agar

Question 26

possible products from M13 cloning and colors of plaques for each?

Answer 26

dna insert present - no b-gal - clear plaque

polylinker present - lacz formed - b-gal - blue plaque

self-ligated - b-gal - blue plaque

Question 27

bacteriophage vector advantages?

Answer 27

can clone large fragments

inherent size selection for large fragments