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Plasmid vectors lecture notes
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Plasmid VectorsCloning vectors:
DNA molecules, derived from naturally occurring plasmids or
bacteriophage, which have themselves been modified to make them more
convenient & versatile
Used to isolate and make large quantities of DNA fragments of interest
Derivatives of Escherichia coli, its plasmids and bacteriophage, are the
principal tools of the genetic manipulator (but other prokaryotes &
eukaryotes can be used as well)
Essence of cloning is to selectively purify specific DNA molecules in a
cloning vector and to amplify it so as to get sufficient quantities of the
recombinant DNA molecules:
- Start with a single E.coli cell containing a recombinant DNA molecule
- Produce a bacterial colony on a Petri dish
- Use colony to inoculate a liquid culture (introduce into a culture
medium)
- Grow overnight
- Purify mgs of recombinant DNA
- Note: PCR can play a similar role
Plasmids = autonomous closed-circular double stranded DNA molecules
found in many prokaryotic cells. Plasmid sizes range from 1kb to over
200kb.
Bacteriophage = viruses that infect bacteria. They consist of a nucleic acid
molecule surrounded by a protein coat. The nucleic acid can be either DNA
or RNA, but the genetic engineer generally uses bacteriophage with DNA
genomes.
Plasmid cloning vectors:
Desirable characteristics:
Small size:
- Easy manipulation: Less vulnerable to damage during purification &
manipulation
- High copy number per cell
- More useful insert per g of recombinant plasmid rather than cloning
vector
- Easier transformation (entering a bacterial cell)
, Selectable marker:
- Can distinguish plasmid-containing from plasmid-lacking cells
- Often antibiotic resistance gene – when grown on agar containing the
antibiotic, only cells containing the plasmid can survive and grow
Range of unique restriction sites:
- Required for ligating DNA fragments into vector. You only want single
sites for each enzyme, so having a range of unique sites means DNA
fragments cut with different enzymes can still be cloned in the same
plasmid vector.
Positive selection/identification of recombinants:
- Selection not only for bacterial cells containing the plasmid cloning
vector, but also for plasmids which contain a cloned insert
High copy number:
- More plasmid DNA per cell
Antibiotic resistance:
Plasmid genes often include antibiotic resistance genes – called selectable
markers
These resistance markers enable the selection of plasmid-containing cells
Pbr322 cloning vector:
p = plasmid
BR = Bolivar & Rodriguez. pBR322 was constructed by B & R from pieces
of naturally occurring E. coli
Has 2 selectable markers (antibiotic resistance genes):
- Ampicillin & tetracycline resistance
- Media containing either of these antibiotics will only permit growth of
plasmid-containing cells
Has a range of unique restriction sites, some within the antibiotic
resistance genes
Positive selection for recombinants via insertional inactivation (cloning into
restriction sites within an antibiotic gene)
Moderate high copy number
Cloning into the bamhi site of pbr322:
Cut Pbr322 with BamHI, with or without removal of 5’ phosphates with
alkaline phosphate
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