TB1-1: Vectors Flashcards
What is a Vector?
a DNA element stably maintained and propagated in a host organism, capable of carrying foreign DNA
What can vectors be based on (i.e. made from)?
Bacterial plasmids
(bacteriophage)
Artificial chromosomes
Eukaryotic viruses
- insects
-mammals
What 4 broad factors should you consider when choosing a vector?
complexity
ease of manipulation
insert capacity
application
What 4 broad types of application do vectors possess (i.e. to think about when choosing a vector)?
Amplification (making lots of it)
Express protein
Express tagged protein (purification or visualisation)
Genome collections/sequencing
Define what a bacterial plasmid is
extrachromosomal small covalently closed circle of DNA in bacteria, carrying gene(s) for antibiotic resistance, conjugation or metabolism of “unusual” substrates
How do plasmid vectors compare in size to natural plasmids?
Smaller than natural endogenous plasmids
How many plasmid DNA copies are produced per 1 genome replication division cycle?
10-100 per cell
What is plasmid copy number?
the average or expected number of plasmid copies per host cell
What is plasmid vector copy number dependent on?
dependent on the rate of initiation of DNA replication
What does MCS stand for in plasmid vectors?
multiple cloning site
What is a multicloning site?
a short region of a (plasmid) vector that contains a range of restriction enzyme recognition sites
What type of cloning does a multicloning site (MCS) allow for?
Directional cloning
What is meant by directional cloning? And why is it necessary?
Inserting an insert in a specific and known direction in a clone. There are two possibilities the insert is cloned in the vector, directional cloning eliminates this doubt.
Important for protein expression
How is directional cloning achieved (as opposed to non-directional)?
DNA insert and vector molecules are digested with two different restriction enzymes to create noncomplementary sticky ends at either end of each restriction fragment. This allows the insert to be ligated to the vector in a specific orientation and prevent the vector from self-ligation.
Name the 4 key features of plasmid vectors
smaller than endogenous plasmids
origin of replication
selectable markers
multicloning site (MCS)
