Cloning Vectors Pgs 94 - 101
Once a gene or part of a gene has been amplified using PCR, the next step in cloning is to insert the DNA into a plasmid or cloning vector so that the fragment can be propagated.
Gene is amplified insert DNA into a cloning vector to be grown. a vector is defined as an organism that transmits an infectious agent from one host to another (Infectious disease definition)
A vector is an agent (such as a bacteriophage or plasmid) that is used to transfer genetic material into a cell. (molecular biology definition) cloning vectors are derived from bacterial plasmids.
Where do cloning vectors come from? Bacterial plasmids!
Plasmids are extrachromosomal DNA:
2000-100,000 bps long most plasmids used in cloning are 2000 – 10,000 bps long.
Bacteria may contain many copies of a single plasmid or single copies of others.
Plasmids are able to replicate independently of the host DNA and most plasmids carry at least one gene. these genes code for a factor or function that helps the bacteria survive
Example: resistance to the antibiotic ampicillin is conveyed by a plasmid carrying an ampicillin-resistance gene.
Plasmids are capable of being transferred from one bacterium to another
This is why plasmids can be used in cloning and why they can be resistant to antibiotics. plasmid designed to clone a gene is different from a plasmid designed to express a cDNA in a mammalian cell line, which is different again from one designed to add a tag to a protein for easy purification.
The primary characteristics of any good vector include:
Plasmids have an origin of replication so they can reproduce independently within the host cell; origin of replication is bacterial which means the plasmid can be replicated by enzymes already present in the host bacteria.
Plasmids origin of replication is what allows them to reproduce independently within a host cell. If the origin of replication is bacterial then the plasmid can be replicated using enzymes that are already in the bacteria.
Bacterial vectors are small (2,000-10,000 bps)
Easy to manipulate
Copy number :
Plasmid is found at specific levels in its host bacterial strain.
High copy number plasmid hundreds of copies in each bacterium.
Low copy number one or two copies per cell.
Cloning vectors derived from certain plasmids have the same copy number range as the original plasmid.
Vectors are usually high copy number
Multiple cloning site (MCS) :
Vectors contain MCS (a series of restriction sites that simplify insertion of foreign DNA into the plasmid).
MCS may have 20+ different enzyme site
Each site unique in MCS and in the Plasmid for each restriction site included in the MCS, the corresponding restriction enzyme will cut the plasmid only at its single site in the MCS
Plasmids can carry one or more resistance genes for antibiotics
If the transformation is successful (if the plasmid enters and replicates in the host cell) the host cell will grow in the presence of the antibiotic.
Common selectable markers are genes for resistance to ampicillin (ampr), tetracycline (tetr), kanamycin (kanr), streptomycin (smr), and chloramphenicol (cmr)
When bacteria are being transformed with a ligation reaction, not all of the religated vectors will necessarily contain the DNA fragment of interest.
To produce visible indicators that cells contain an insert :
Vectors contain reporter genes (distinguish them from cells that do not have inserts)
Common reporter genes are -galactosidase (-gal) and Green Fluorescent Protein (GFP)
Newer plasmid vectors use positive selection, in which the inserted DNA interrupts a gene that would otherwise be lethal to the bacteria
If foreign DNA is not successfully inserted into the MCS, the lethal gene is expressed and transformed cells die.
If the foreign DNA is successfully inserted, the lethal gene is not expressed…