Vectors II Flashcards
(27 cards)
What are fusion proteins?
fused genes for 2 proteins
What are the uses of fusion proteins? Describe
Identification
- antibody binding
- fluorescent
In quantification
-surrogate proteins
In purifications
-tag proteins bind to a particular substance
List some examples fro
hexahistidine - high affinity to Ni chitin Arg-tag S-tag MBP CBP Amylose
What are the 3 types of plant vectors?
based on natural occuring plasmids from Agrobacterium - Ti, Ri
direct gene transfer - various plamid
plant viruses - caulimo, gemini
What happens in an A. tumefacians infection?
Ti-plasmid > T dna integrated into plant dna > tumors >. synthesize opines
Show how Ti-Plasmid is used in plant cloning
ss
Why phage vectors are better over plasmids? drawbacks?
plasmids have to transport. cant do big pieces but phages inject and propagate. infection is efficient than transformation
more complex
depend on bacteria
Virus structure types?
head with tail - lambda
filamentous - M13
Viral life cycle types?
lytic
lysogenic
main steps of pattern of viral infection?
infection
replication
phage formation
lambda phage properties? role of cos sites?
package
rolling circle replication
Show how dna is packaged inside lambda phage
cos- concatamer
Show how dna is replicated in lambda phage
dna injected
concatamer formation
head proteins + endonuclease A expressed
cuts dna @ cos
package into capsids
What is the drawback of lambda phage as a vector? How is it minimized?
capsid – physical constraint on amount of DNA
by remove unnecessary parts and make designer vectors
Draw & give exampls for insert. & subs. vectors
Lgt10
Charon 16A
Embl3
Embl4
how a foreign gene is cloned into lambda phage vector?
t4 dna ligase
how to do in-vitro packaging of a lambda vector? explain in steps
2 methods?
READ MORE IN-VITRO PACKAGING!
READ!
M13 phage characteristics?
ss
What are the advantages of M13?
ds repli. form
ss during the infection
Visualize the genome of M13 phage
ss
Explain the life cycle of M13
ss
Show how vectors are designed from M13 phage
m13mp1 m13mp2 m13mp7 m13mp8 m13mp9
How to use M13 in cloning? explain with steps
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
