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Mobilizing DNA: vectors for

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

PLASMID REPLICON COPY #

pBR322 pMB1 15-20

pUC Modified form of 500-700


pMB1 (RNAII
mutation)
pACYC p15A 18-22

pSC101 pSC101 about 5


Plasmid copy number
High copy number plasmids
Workhorses of molecular cloning
Used for almost all routine manipulation of small
(<15 kb) recombinant DNAs

Low copy number plasmids


For genes that are lethal or unstable in high copy
number plasmids
For constructing Bacterial Artificial Chromosomes
(BACs) that can propagate large (>100 kb)
recombinant DNAs
Replicon -- how the plasmid replicates

Governs replication of plasmid and number of plasmid


copies per cell (copy number)
A replicon includes:
origin of replication (ori: a site on the DNA)
associated factors
> 30 different replicons known, but most plasmids
used today have pMB1 (or the close relative colE1)
replicon
pMB1/colE1
replication
mechanism

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

CAUTION: Over time beta-lactamase is secreted


into the medium where it breaks down the
antibiotic and depletes it. Eventually this allows
the growth of ampicillin/ carbenicillin sensitive
cells, defeating the selection
Antibiotic resistance genes
Chloramphenicol acetyl transferase (CAT): inactivates
chloramphenicol (cm), which normally inhibits peptidyl
transferase activity of the ribosome (no protein
synthesis = dead cell)

Another use for cm:


replication of plasmids with pMB1/colE1 replicons is not
inhibited by cm
Cm-treated cells stop growing but continue making these
plasmids, this is a way to amplify plasmid copy numbers
prior to a plasmid prep
Antibiotic resistance genes

Tet A (C ) protein: confers resistance to tetracycline (an inhibitor of protein


synthesis) by pumping this antibiotic out of the cell

Bacterial aminophosphotransferases: confer resistant to kanamycins


(aminoglycoside antibiotics that inhibit protein synthesis) by transferring
the gamma phosphate of ATP to a 3 hydroxyl group of the kanamycin
The ideal plasmid

1. Confers a readily selectable phenotypic trait


2. Has single sites for many restriction enzymes
3. Low molecular weight
-- Gives higher copy #, stability, and transforming
efficiency
-- Can accept larger pieces of DNA
-- Easier to handle (less susceptible to breakage)
pBR322
The first widely useful cloning vehicle

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).

But: pBR322 has low


copy number, large size,
and too few options for
cloning sites
Boldface indicates the restriction site is
present in only one site within the plasmid
pUC plasmids
second generation cloning vectors

Reduced size (about 2000 bp)


Multiple cloning site (MCS, also called poly-linker):
unique sites for lots of different restriction enzymes
Very high copy number (mutation in RNA II)
New blue-white screening tool for recombinants
(alpha complementation is disrupted by foreign DNA
in the MCS)
Alpha complementation
Plasmid encodes N-terminus of X-gal
beta galactosidase (alpha
fragment)
Host strain encodes the C-
terminus of beta galactosidase
(omega fragment)
Beta galactosidase function is
only seen in the presence of both
the N- and C-terminal fragments
Beta gal function can be
monitored by the cleavage of X-gal
which yields a bright blue product
(blue colonies on a plate)
Bright blue
An alpha complementing plasmid vector

(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

Vectors containing bacteriophage RNA polymerase promoters:


for production of a specific RNA (probe synthesis, in vitro
translation, etc.)
Low copy number vectors: for cloning of unstable or toxic
genes
Vectors designed for expression of specific proteins (for further
purification and biochemical characterization). Proteins may be
synthesized with tags to assist in purification
Transformation of E.coli
with plasmid DNA

E.coli strain: must be antibiotic sensitive, best if it lacks


restriction-modification systems
Make cells take up DNA by
Chemical competence
Electroporation
(natural competence--not E.coli though)
Chemically competent cells-
basic method

Grow cells to A600 of 0.4, spin to get cell pellet


Resuspend cells in CaCl2 (100 mM), pellet again
Resuspend in small volume of CaCl2/glycerol
Freeze cells (-80C) or go straight to
transformation protocol
Transformation of chemically
competent cells
DNA binds to cells Mix DNA and competent cells, on
ice for 30 min.
DNA uptake by cells Heat shock (42C) for 1.5 minutes
Cells recover Add growth media, 37C for 1 hour
Plate on growth medium plus
Selection occurs selection (antibiotic) for the
plasmid

Efficiency ~ 106 - 107


cells/microgram plasmid DNA
If cells are good:
Transformation by electroporation
> 109 transformants/microgram DNA (ideally)
Grow cells to A600 of 0.4
Centrifuge and resuspend in water + 10% glycerol (do this
4 times to reduce conductivity)
Place cells with DNA in electrode-containing cuvette,
deliver electrical pulse
If there is arcing (sparks) transformation efficiency will be
poor (uneven transfer of charge). To avoid this make sure
the ion concentration is very low (less than 10 mM salt)
When cloning a piece of DNA consider:
1) Choice of vector: what kind of plasmid vector to use
(which restriction sites can be used in the vector)?

2) Ligating DNA to vector: how will the ligation reaction be


set up to facilitate getting what you want?

3) Moving DNA by transformation: what strain of E. coli will


you transform into? Which method for transformation?

4) Screening for successful ligation products (recombinant


plasmid DNA): how will the recombinant plasmids be
identified?
Setting up a transformation--how will
the competent cells be treated?
1. No plasmid (negative control, nothing should
grow on this plate)
2. Supercoiled plasmid of a known concentration
(to determine efficiency of competent cells)
3. Vector DNA (dephosphorylated?) ligated
without insert DNA (background
transformants)
4. Vector DNA ligated with insert DNA (desired
products)
Example outcome of a successful
transformation: chemically competent cells

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

Upper limit for clone DNA size is 12 kb


Requires the preparation of competent host
cells

Inefficient for generating genomic libraries as


overlapping regions needed to place in proper
sequence
Preference for smaller clones to be transformed
If it is an expression vector there are often
limitations regarding eukaryotic protein
expression