Vectors Flashcards
Episomal
Non-intergrated
Why is DNA transported in plasmids?
Separate from chromosome so can move
Free DNA is attacked by cells
When does ampicillin kill bacteria?
When they’re dividing
F-plasmid genes
Cause bacteria to make sex pili proteins
Attaches to new cell, form cytoplasmic link
Mobilisation proteins move plasmid DNA through link so plasmid present in both cells
Copy number (vectors)
How many copies can be supported
What does a relaxed origin of replication mean?
High copy number
Generally small plasmid
Why do plasmids have marker genes like those for antibiotic resistance?
To detect if the bacteria has taken up the recombinant plasmids
Bacteriacide
Antibiotic that kills bacteria immediately
Bacteriostatic
Antibiotic that kills bacteria when division occurs
Beta-lactomase
Enzyme coded for by ampicillin resistant gene
Cleaves B-lactam
What are important traits required by plasmids to be used in cloning?
dsDNA Relaxed origin of replication for lots of copies Have 1 or more selectable markers Has restriction enzyme sites Suitable promoter
Why does pBluescript have 2 origins of replication?
1 normal
1 from filamentous bacteriophage
Lambda
Large virus
Infects E.coli
Linear dsDNA inside rigid geometric head
How can we use lambda as a cloning vector?
Delete a non-essential gene cluster and replace it with cloned sequences (up to ~20kb)
Can then be injected into cell by lambda
Cos sites
Lambda virus genome has short complimentary ssDNA ends/overhangs
Can allow the genome to circularise
What lambda enzyme digests cos sites?
Virus endonuclease
Why would you want to digest cos sites?
So the circular vector can be linearised
Much superior method of gaining entry into cells
Cosmid vector
Vector that has cos sites from lambda virus
BAC vectors
Bacterial artificial chromosomes
Largest capacity vector
Low copy number
Why is there no need to include a selectable marker with retorviruses?
They integrate their DNA straight into the cells DNA
Baculovirus
Insect specific virus
Strongest gene promoter
Polyhedrin protein made to very high levels in infected cells
How are baculoviruses used as vectors?
The gene of interest replaces polyhedrin coding sequence
What factors should be considered when choosing a vector?
Size of fragment to be cloned
Goal of cloning
Target cells or organism
Suitability of vector
Gene library
Large collection of individually cloned DNA fragments that represent all genetic sequences of an organism, cell or tissue sample
What is the ideal gene library like?
As small as possible
As small fragments as possible
Expression vectors
Contain necessary regulatory sequences
Fusion peptides
Additional short DNA sequence in vector can fuse a novel peptide onto a recombinant protein
What can fusion peptides do?
Stabilise the protein and prevent degredation Solubilise the protein Direct protein targeting Allow protein detection Allow protein purification
What can high levels of recombinant protein translation result in?
Inclussion bodies
Inclussion bodies
Aggregates of insoluble protein
Caused by high levels of recombinant protein translation
Partial restriction digest
Use an enzyme that cuts DNA sequence frequently
Stop halfway through
Results in a random distribution of cut sites
What agent removes a cleaves methionine?
Cyanogen bromide
How do fusion peptides allow recombinant protein purification?
Ligans fixed to inert surface
Pour cell lysate through column and tagged proteins bind to ligand
Release bound fusion protein
