Plasmid Vectors
Plasmid Vectors
Plasmid Vectors
propagation in E. coli
Plasmids
Bacteriophage
M13
Lambda
Cosmids and BACs
Plasmids and transformation
I. Properties of plasmids
II. Plasmids as cloning vehicles (vectors)
III. Ligation and transformation, and
identification of recombinant plasmids
Plasmids
Extrachromosomal, double-stranded, usually
circular, supercoiled DNA molecules
Found in many bacterial species
Replicate and are inherited independently of the
bacterial chromosome
Maintain copy number in cell through an origin of
replication (replicon)
Usually have genes coding for enzymes that provide
benefits for the host bacterium, eg. antibiotic
resistance
a generic, minimal plasmid
restriction site
antibiotic for cloning
resistance
pBi430/530
1500 base pairs
(a manageable size
origin of
replication
Common plasmids and their stats
1
Deletion of Rop or
mutation of RNA II
2 cause increases in
replication and
copy number
4
Plasmid maintenance
Plasmids contain selectable markers: genes
carried by the plasmid that confer functions
required for host survival
Selection: only those cells with the plasmid will
survive
Allows transformation (a rare event) to be feasible
A way to keep cells from losing plasmids that may
otherwise confer a selective disadvantage
Antibiotic resistance genes
Beta lactamase (bla): breaks down ampicillin
and carbenicillin (inhibitors of cell wall
synthesis). Cells carrying this gene are often
termed ampr
Created using
transposition and
restriction/ligation
reactions
Utility of pBR322:
Clone into sites in the Tcr
gene, which allows
identification of
recombinants--these will
be amp resistant but tet
sensitive (initially plate
on ampicillin, then
pBR322 replica plate on
tetracycline plates).
(MCS)
pUC 19
DNA in the MCS interrupts the lacZ gene (no Beta galactosidase)
Alpha complementation
Plasmid encodes N-terminus of beta galactosidase
(alpha fragment), with an MCS
Foreign DNA in the MCS, no alpha fragment
No alpha fragment, no -gal
No -gal, no blue color (white colonies)
Colony without
foreign DNA in MCS
pUC19
transformation plate Colony with foreign
DNA in MCS
Third generation cloning vectors:
specialized plasmids
1) No DNA--No colonies
2) 2 nanograms (10-9 g, 10-3 micrograms) supercoiled
plasmid DNA--500 colonies (efficiency of cells: 2.5
x 105 transformants per microgram DNA)
3) Vector alone--small number of colonies
4) Vector plus insert--larger number of colonies than
for #3
Identifying recombinant
plasmid-containing cells
Alpha complementation: most white colonies represent
presence of insert DNA blocking functional beta
galactosidase
Increase in number of transformants in presence of
insert vs. absence of insert
vector treated with alkaline phosphatase
Directional cloning--preventing religation of vector
Must screen colonies/plasmids for inserts, usually by
PCR
Confirm clones by sequencing
Colony hybridization to screen the
bacterial plasmid library:
1. The cDNA library (bacteria
containing different cDNAs) is
plated on agar plates in
appropriate media.
2. A nitrocellulose paper is pressed
upon the the bacterial colonies.
Some bacteria are transferred to
NC paper.
3. The NC is treated with alkali to
lyse the cells and expose the
cDNAs.
4. The DNA binds to NC paper, and
a radioactive DNA probe
corresponding to the desired
gene is used to hybridize with
the NC paper.
5. DNA from the Colonies with the
desired gene will be seen on X-
ray film after the exposure of the
hybridized NC paper.
The Major Limitation of Cloning in Plasmids