Download Setting up a transformation--how will the competent cells be treated?

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, in
transformants/microgram)
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
– Insert treated with alkaline phosphatase
– Directional cloning--preventing religation of vector
– SCREEN colonies/plasmids for inserts, usually by
PCR
Confirm clones by sequencing
Mobilizing DNA: vectors for
propagation in E. coli
•Plasmids
•Bacteriophage
 M13
 Lambda
•Specialized cloning vectors
 expression vectors and tags
 vectors for large pieces of DNA,
e.g. Cosmids and BACs
Bacteriophages: useful vectors in
molecular cloning
I.
II.
Lambda: a “head and tail” phage
--The lambda life cycle
--Basic cloning in lambda
M13: a filamentous phage
--Life cycle
--genome structure
--phagemids
Bacteriophages
• Viruses that infect bacteria
a) “head and tail”
b) Filamentous
c) etc….
• Nucleic acid molecule (usually DNA)
– Carrying a variety of genes for phage replication
– Surrounded by a protective protein coat (capsid)
• Infection (instead of transformation):
– Phage attaches to outside of bacterium, injects DNA
– Phage DNA is replicated
– Capsid proteins synthesized, phage assembled and released
Bacteriophage lambda
• “head and tail” phage, very well-studied
• Large, linear genome--48.5 kb
– Central region of genome (“stuffer”) is
dispensable for infectious growth--it can be
engineered out
• Two lifestyle modes
– Lytic: replicative mode
– Lysogenic: latent mode
• Useful for cloning 5-25 kb DNA fragments
lambda genome
Lambda: lytic infection
Linear DNA
decision
Lambda: latent infection (lysogeny)
Lysogen: an E. coli
strain that can be
made to lyse under
the right conditions
(e.g. UV treatment)
Lambda as a cloning vector
• Insertional vectors (clone into single
restriction site, can only increase genome
size by 5% (size of foreign DNA insert
depends on the original size of the phage
vector, about 5 to 11 kb)
• Replacement vectors (removing “stuffer”), can
clone larger pieces of DNA, 8 to 24 kb
(sufficient for many eukaryotic genes)
Cloning in lambda phage--an overview
Left arm
“Stuffer”
Right arm
1) Restrict, purify right and left arms
2) Ligate with foreign DNA
3) “Package” ligation mixture into phage heads
4) Plate mixture on E. coli, individual plaques
represent recombinant clones
Examples of “replacement” lambda vectors
How to transfer
recombinant lambda
into cells?
The packaged
phage particles
are infectious
Selecting recombinant lambda phages I
• There is a minimal size of DNA that can be
packaged in lambda phage heads
• Remove stuffer (for some replacement
vectors), the ligated “arms” cannot be
packaged without an insert present
• Selection: only thing that is infectious is the
recombinant DNA product
Selecting recombinant lambda phages II
• Wild type lambda cannot grow on E. coli
infected with phage P2 (spi, or sensitive to P2
inhibition), spi+ conferred by red and gam
genes in “stuffer”
• Only phage lacking stuffer (they don’t have
spi gene) can make plaques on lawn of E.
coli containing a P2 lysogen
Filamentous phages: M13
• Single-stranded, circular genome, 6.4 kb
• Can clone pieces of DNA up to 6X the M13 genome size
(36 kb) -- but the larger the DNA, the less stable the clone
is…..
• Useful for
– Sequencing
– Site-directed mutagenesis (later)
– Any other technique that requires single stranded DNA
• Drawback: foreign DNA can be unstable (slows down host
cell growth, so deletions confer a selective advantage)
M13 structure
Used in
‘phage
display’
techniques
ss
M13 life cycle:
an overview
ds
ss
Isolate for cloning
M13: life cycle
Cell has to have
F plasmid for
infection to
work
M13:
life cycle
Isolate phage
(and singlestranded DNA)
in supernatant
Isolate
doublestranded
DNA by
standard
plasmid
prep
M13 doesn’t lyse cells, but it does slow them down
“lawn” of
E. coli
M13 infections form plaques, but they are “turbid”
M13 mp18: engineered for alpha complementation
Phagemids: plasmid/M13 hybrids
• Plasmids containing both plasmid (colE1) origin and
bacteriophage M13 origin of replication
•To recover single-stranded version of the plasmid (for
sequencing, e.g.), infect transformed (male) strain with a
helper phage (M13KO7)
• Helper phage cannot produce single stranded copies of
itself, but provides replication machinery for single-stranded
copies of the phagemid DNA
• Phagemid single stranded DNA is packaged and extruded
into supernatant--can then be isolated for sequencing, etc.
Uses of Bacteriophages:
Lambda -- large-ish DNA fragments
•for gene cloning (large eukaryotic genes)
•Excellent selection capability (stuffer stuff)
•Clone lots of precisely-sized DNA fragments
for library construction
M13 -- single-stranded DNA
•Sequencing
•Site-directed mutagenesis
•Etc.
Specialized vectors for E.coli
I.
Expression vectors
II. Large DNA molecules: Cosmids, PACs,
and BACs
Course packet: #25, 26, 27
Expression vectors
• For production of specific RNA or protein of
interest
• Optimized for transcription, translation, and
post-translational handling
Typical expression vector cloning site:
promoter
MCS
tags tags
Transcription
terminator
Expression vectors:
RNA: expression
occurs in vitro
(purified plasmids)
Making micro RNAs for RNAi: one example
How to control transcription driving
RNA/protein expression in vivo?
• T7 RNA polymerase promoters: T7 RNA polymerase
under control of lac repressor (induced by IPTG)
• Lambda PL promoter, controlled by lambda repressor
(which is regulated by trp repressor)
• pBAD promoter, controlled by the araC protein in
response to arabinose
pET vectors: protein expression
Helper tags for protein
production and purification
• 6/7 histidine tag: interacts very specifically with
Ni2+ ions, which can be immobilized on columns or
beads
• Biotin carboxylase: covalently attaches to biotin,
biotin binds to streptavidin which can be
immobilized on columns or beads
• Epitopes (e.g. c-myc) for specific antibodies can
be included as tags--purify on antibody column
• Tags can be engineered to be removable
high affinity,
high specificity
Using tags in protein purification
A protein purification scheme--removable tag
Cloning large DNA fragments
• Cosmids: bacteriophage lambda-based
• Bacteriophage P1 plasmids
• BACs: F plasmid-based
replicon
Lambda
colE1
P1
P1
F
ARS
transfer
transfection
transfection
transfection
electroporation
electroporation
transformation
This is a very good table to be familiar with
Why clone large pieces of DNA???
Make libraries: genome broken up into small,
manageable, organizable pieces
Each recombinant DNA fragment from the ligation--a
piece of the genome
How many recombinant DNA molecules are required in a
library to get complete coverage of a genome?
N=
ln(1-p)
ln(1-f)
P = probability of getting a specific
piece of the genome (1.0 = 100%)
f = fractional size of clone
DNA relative to genome
N = number of clones needed
99% probability of having a given DNA sequence
17 kb fragment library
Mammalian genome: 3 x 109 base pairs
ln(1 - 0.99)
N=
(
ln 1 -
1.7 x 104
3 x 109
)
N = 8.1 x 105 clones required
Cosmids:
• 5 kb plasmids, antibiotic resistance, plasmid origin
of replication
• Contain lambda cos sites required for packaging
into lambda phage heads
• Packaging only occurs with 37-52 kb fragments-selection for large fragments
• Packaged DNA is inserted into cells and then
replicates as a very large plasmid
Cloning in a cosmid
Desired ligation
Products--these
are packaged
Cloning in a cosmid
Instead of
transformation, desired
ligation products are
packaged and then
transfected into cells
Selection for colonies,
not screening of
plaques (not infectious)
Cosmids: a specific cloning scheme
split
Prevents
ligation without
insert
Sau3A:
GATC
5’ overhang
(compatible
with BamHI
sticky end)
Prevents
multiple
fragments
Phage P1 vectors: cloning up to 100 kb DNA fragments
85-100 kb
Phage P1 vectors: cloning up to 100 kb DNA fragments
Efficiency of packaging is typically low: thus it is not
good for making large genomic libraries
Phage P1 vectors:
cloning up to 100 kb DNA fragments
PACs: like P1 vectors but the DNA is not
packaged (transfer by electroporation)
BACs: Bacterial Artificial Chromosomes
• Based on the F factor of E. coli:
--100 kb plasmid, propagates through conjugation
--low copy number (1-2 copies per cell)
--2 genes (parA and parB): accurate partitioning during
cell division
• BACs: just have par genes, replication ori, cloning sites,
selectable marker
• Can propagate very large pieces of DNA:
•up to 300 kb
• Relatively easy to manipulate: move into cells by
transformation (electroporation)
General BAC vector
Cloning, etc
selection
7 kb
replication
o---- Cloning strategies ---o
I.
II.
Making DNA “libraries” (from genomic DNA, mRNA
“transcriptome”)
Screening to identify a specific clone (the needle in
the haystack)
-- by the sequence of the clone
-- by the structure or function of the expressed
product of the clone
Course reading: #28
(and 29)
Overview of strategies for cloning genes
1)
2)
3)
Get DNA
Ligate to vector
Transform or transfect
Look for the gene…
4)
1) Get DNA
Genomic DNA
RNA
Ligate to vector: how to make this reaction favorable?
This yields a “library”, a representative set of all the
pieces of DNA that make up a genome (or all the cDNAs
that correspond to the “transcriptome”)
cDNAs from different tissues reflect the different RNA
populations that you find in distinct cell types:
Hence “liver” vs. “brain” vs. “heart” cDNA libraries
There are lots of ways to identify a particular gene…
Overview of strategies for cloning genes