FIRST CLASS Lecture notes Cell And Molecular Biology

Plasmid Vectors
Cloning 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