Plasmid DNA

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Plasmid DNA
Density gradient centrifugation in CsCl
ethidium bromide

Agarose gel electrophoresis. Detection by
staining with ethidium bromide.
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Plasmids independent genetic elements found in
bacterial cells.
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l
Antibiotics antibiotic resistance 1) is the
cell sensitive? - cell wall/membrane
permeability? - sensitive target present? 2)
field of action - cell wall synthesis -
membrane functions - replication -
transcription - translation 3) resistance
mechanisms (nb. sometimes different
mechanisms are possible with the same
antibiotic) - prevent penetration of the cell
keep the cell antibiotic-free - degradation
of the antibiotic remove it (intra- or
extracellularly) - modification of the
antibiotic inactivation of the agent -
modification of the target prevent
the activity at the target - amplification of
the target titration of the agent gt
tolerance by excess of target

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l
Antibiotics antibiotic resistance Resistance
mechanism (nb. sometimes different
mechanisms are possible with the same antibiotic)
- prevent penetration of the cell vb.
tetA TcR pump out - degradation of the
antibiotic vb. bla ApR in periplasm or
extracellularly - modification of the antibiotic
vb. cat CmR inactivation of the agent -
modification of the target vb. rpsL
(ribosomal SmR streptomycin-insensitive
mutant protein) aph SmR inactivation
by phosphorylation) - amplification of the
target vb. 23S rRNA tsR thiostrepton
insensitive ribosomes (methylation of
nucleotide in )
Tc (tetracyclin) translation-inhibition 30S
ribosomal subunit binds the 16S rRNA and
blocks the aminoacyl-tRNA Ap
(ampicillin) inhibition of peptidoglycan
synthesis Cm (chlooramfenicol) translation
inhibition 50S ribosomal subunit
Sm (streptomycin) translation inhibition 30S
ribosomal subunit ts (thiostrepton) translation
inhibition 50S ribosomal subunit
tetA gene encodes an antiporter system bla gene
encodes a b-lactamase hydrolyseert een b-lactam
ring cat gene encodes a chlooramphenicol acetyl
transferase modifies the antibiotic rpsL gene
encodes the S12 protein of the small ribosomal
subunit thiostrepton resistance by methylation
of the 23S rRNA
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The Escherichia coli origin of replication

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The ori of plasmid pSC101

R1, R2 and R3 are 3 iteron sequences where RepA
binds and initiates replication, but at high
plasmid concentration it also makes an
intermolecular (handcuffing) between 2 plasmid
molecules and then inhibits further replication.
repA is the sole plasmid-encoded gene required
for replication. RepA autoregulates its own
synthesis by binding to the inverted repeats IR1
and IR2. DnaA binding sites are indicated, as
well as the par locus.
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Restriction and functional map of plasmid
pSC101. The 7 HincII fragments are named A to
F. IS101 and IS102 are 2 insertion
sequences. oriT is required for mobilisation by
conjugative plasmids. oriV lies in
replication functions. par is the region
required for stable partition of pSC101. TcR
tetracyclin resistance gene

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Plasmid ColE1
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Origin of replication of ColE1 plasmid

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Properties of (current) general-purpose plasmid
vectors (2nd generation vector) - modular -
limited size (the smaller the better") -
unique cleavage sites gt different kinds of
ends, different places (outside ori) -
phenotypical markers selection and/or
identification (screening) 2 selection markers
gt 1 for insertional inactivation or 1
selection marker an identification marker for
screening or 1 negative selection
marker for the vector a positive selection
marker for recombinants - extra properties for
general use (sequence analysis, mutagenesis,
etc.)

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ColE1 has suitable characteristics, if better
markers can be introduced - ori requires
polymerase I (polA) and RNaseH - does not
require translation - ori RNA II, RNA I, Rop
deletion of rop causes increase of copy number
- colicin E1 production, colicin E1 immunity
replaced by AB-resistance genes - special loci
par, cer - par of ColEI is deleted in the
first derivatives - if there is more than 1 cer
sequence per plasmid, the cell Xer protein
promotes a site-specific recombination
(resolution). - is present in the cell at a
large copy number (gt 15 to 50 or more depending
on the author) gt advantagous for DNA analysis

construction of pBR322 sequenced by G.
Sutcliffe - three segments from different
sources - completely sequenced construction
was not planned as such but basically followed
from a diversity of genetical and biochemical
studies/manipulations.
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pBR322
4361 bp
PstI EcoRI BamHI SalI EcoRV HindIII ClaI
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Construction of pBR327 from pBR322

"Breathing" at the ends of the linear DNA led to
extra deletions of 10 (left) and 14 bp (right),
respectively.
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Conventional zero point on the (circular) map of
pBR322 is the central position in the EcoRI site
the first T in GAATTC is position 1. Hence
the second A is positie 4361.
The combination of two resistance genes in
pBR322 limits the possibilities of consecutive
manipulations (after insertion in one gene, the
second one remains active) Moreover, cloning in
strains that are resistant to one of the
antibiotics is difficult (e.g. by the presence
of a transposon). There was a need for more
combinations. At the same time, a compatible
replicon could be used, so that a single cell
could harbour it together with a
pBR322-derivative. Such replicon was found with
the cryptic plasmid p15A, whose ori is similar to
the one of ColE1 (but is compatible). Thus,
pACYC177 kanamycin resistance (KmR)
ampicillin resistance (ApR) pACYC184
tetracyclin reistance (TcR) chlooramfenicol
resistance (CmR) were developed.
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ori ?
ori
pACYC177 3941 bp
pACYC184 4245
bp
pA15 replicon compatible with ColE1
replicon
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Further developments Decreasing the size of
the vectors by deletion of non-functional
regions. Introducing "multiple cloning sites"
(MCS) or polylinkers (see
below) Introducing markers for "positive"
selection
Vectors for "positive" selection of recombinants
some examples
Conditions can be created at which an E. coli
cell hosting a plasmid vector does not survive if
it harbours or expresses a particular marker.
Insertion in responsible marker DNA then allows
"positive" (i.e. direct) selection of the
recombinants. Nevertheless, it may not be
forgotten that the vector DNA itself should be
prepared as well hence, a growth condition is
also required at which the cell survives in the
presence of an intact vector. If such
conditions are prepared by addition of a
particular substrate to the medium, it may be
sufficient to omit this substrate in order to be
able to prepare vector DNA.

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Other examples sacB (SacB is levan sucrase
it hydrolyzes sucrose to levan that accumulates
in the periplasm and is lethal to E. coli)
ccdB encodes a topoisomerase II inhibitor,
which induces the SOS pathway and leads to cell
death of E. coli if CcdA is absent. The ccd
operon is encoded on the F plasmid.
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cI is the l repressor gene, its promoter is PRM
(see Bacteriophage l vectors). PR is another l
promoter which is under control (repression) of
CI. The promoter of the TcR gene has
been removed, so that expression of the
tetracyclin resistance depends on the absence
of an active CI. The latter is the result of
insertion in BclI or HindIII.

BclI is compatible with all 5 GATC protruding
ends. (But unfortunately inhibited by Dam
methylation.) The limited availability of
suitable restriction sites is a limiting factor
for the use of this kind of vectors. (See also
other examples).
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The rpsL gene is also named STRS. The protein
it encoded (S12) is the target of streptomycin
and hence causes the sensitivity of E. coli for
this antibiotic. (S12 is a protein in the
small subunit of the ribosome, and thus required
for functional translation.) The wt-rpsL is
cloned on a plasmid, and is dominant over a
mutant rpsL on the chromosome of the host cell.
The plasmid vector turns the host sensitive to
streptomycin, wherease insertion in SmaI, HpaI
or SstII restores resistance. Only recombinants
survive in the presence of streptomycin.

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Similar to the STRS system, with an analog of
phenylalanine replacing streptomycin. The
target is the a-subunit of the E. coli
phenylalanine-tRNA synthetase. This subunit is
encoded by the pheS gene, and is sensitive for
to analog p-fluoro-phenylalanine. The mutant
PheS12 is resistant against the analog and
dominant over PheS. Insertion in PstI (3
extension) or HincII (blunt).

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regulated by l CIts
promoter PL or PR
plasmid pEcoR251 ori origin of replication Apr
ampicillin resistance gene ecoRI-endo EcoRI
endonuclease gene Expression of this gene causes
synthesis of EcoRI endonuclease and destroys
all DNA, thus killing the cell. The promoters
PL or PR are switched off by active l repressor
(CIts) at 32C but induced at 42C. Insertion
in HindIII, BglII or PstI.
ecoRI-endo
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In pGV450, selection is based on type I
restriction by providing the restriction site for
insertion in the center of the EcoK recognition
site. There are 6 base pairs between the two
sides of the recogntion site. A A C N N N N
N N G T G C EcoK recognition site A A C G G
A T C C G T G C insertion of BamHI fragments
(or other fragments with 5'-GATC
extensions) A A C C C C G G G G T G C
insertion of blunt end fragments in SmaI
Limitations - there is no full selection but
only "restriction" of survival (like in
natural restriction-modification systems) -
there is one chance in 100 that the recognition
sequence is restored (depending on the flanking
sequences of the cloned fragments) Analogously
ecoB strains may be used T G A N N N N N N N N
T G C T
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General remarks - the use of positive
selection for recombinants is often rather
limited, especially because of the limited
number of appropriate restriction sites in the
target gene. - the markers are more often used
as (intact) selection modules. - a marker is
not always either a positive or a negative
selection marker, but may be used as either one,
depending on the growth and environmental
conditions example (with cloning in yeast)
URA3 - a yeast URA3- mutant
survives in the presence of uracil in the growth
medium or can be complemented by a plasmid
expressing the wt-URA3 use for negative
selection - addition of 5-FOA
(fluoro-orotic acid) to the growth medium kills
URA3 cells (by incorporation of the analog)
positive selection
MCS ('multiple cloning site') or polylinker
When vectors became smaller and smaller, and it
became easier to detect recombinants by size on
an agarose gels, often a short, synthetic region
was built in in the vectors so that a large
number of "unique" restriction sites, close to
each other, became available for further
manipulation. Exceptionally, such MCS could
be constructed within a coding region of a gene
(the best known example is lacZ and lacZa (see
below, a.o. M13 and pUC vectors and
derivatives).
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Minimizing vector size was achieved by indirect
control on selection, by using amber mutants of
the target gene. In the PiAN vectors, a
suppressor tRNA gene is provided (occupies less
than 150 bp space (in principle, together with
the 600 bp ColE1 ori, the vector may be as short
as 750 bp). SupF (tyrT) inserts a tyrosine at a
codon that is mutated to UAG. (With SupE
(glnV) this is an asparagine.)

Application cloning of a target for homologous
recombination (in the polylinker) gt selection
of cells in which PiAN-construct is inserted (by
recombination) and thus brings the suppressor
phenotype with it.
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Example phenotype of a host cell depends on
the presence of PiAN (or pVX) E. coli W3100
(p3) KmR (ApS and TcS the resistance
genes are present on p3 amber mutant, i.o.w.
UAG mutation(s) in an essential coding region
only KmR is present as "wild-type" on p3)
this may also be shown as Apam en Tcam
E. coli W3100 (p3 PiAN) KmR, ApR, TcR
the variants of the latter resistances are
produced by suppression of the UAG triplets by
the suppressor tRNA encoded by PiAN.
(Eventual Tyr or Gln substitution should make
active proteins!)
See also later screening of genomic libraries.
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Markers for identification or screening If
transformants can be selected by an appropriate
marker, it may be sufficient (and interesting)
to make a direct discrimination between vector
transformants and true recombinants by relying on
a visible (phenotypical) characteristic of the
colonies. In vectors of filamentous phages
(e.g. M13), such an approach was realized using
the E. coli lac operon, in particular its lacZ
gene, encoding the b-galactosidase. Since the
lacZ gene is far too big, a fragment (lacZa) can
be used instead, if suitable complementation
activity is provided by the host. (Known as
a-complementation). (See at the "Bacteriophage
vectors" for more details.) The lacZa region
was substituted for the tetA region (and adjacent
DNA) of pBR322, and created the pUC-vectors. In
the coding part of the N-terminal region LacZa,
mutations and/or extra DNA segments (MCS) are
added bearing unique restriction sites.
Interruption in one of these leads to
inactivation of the complementation and of the
colored phenotype. The vectors are
substantially smaller than pBR322 (sizes around
2500 bp). Selection relies on ampicillin
resistance. Identification (difference between
vector and recombinant) on color. The copy
number is also increased by effects onto ori
(a.o. removal of the rop gene and the region
encompassing nic/bom (oriT)).

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b-galactosidase is a tetrameric protein each
monomer (116 KDa) is 1021 amino acids
long. There is an EcoRI a few triplets from the
right end of lacZ (i.e. close to the C-terminus
encoding region) Intracistronic complementation
a-peptide region W region gt
both segments are coded onto a different genetic
element, but the products of both interact and
make active enzyme e.g. the W-segment on
the chromosome and the a-peptide segment on a
plasmid or phage The DNA coding for the
a-peptide has no EcoRI site. But it was created
by mutagenesis at the 5th codon
(start)-Thr-Met-Ile-Thr-Asp-Ser-Leu-... atg-ACC-A
TG-ATT-ACG-GAT-TCA-CTG-... wild-type atg-ACC-ATG
-ATT-ACG-AAT-TCA-CTG-... mutant ................
...-Asn-........... EcoRI Into this
EcoRI site different polylinkers were inserted
if polylinkers are a multiple of 3 bp, the
a-peptide activity may be preserved. (Must be
checked for each vector construct)
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• lacZa was transferred in a modular fashion from
  the filamentous phage vectors to pBR322.
• lac operon, lacZ, lacZa
• lacZDM15 and a-complementation
• E.coli Dlac-pro
• lacI, LacI, titration and lacIq
• bleu-white identification inductors,
  substrates IPTG, BCIG
• IPTG iso-propyl thiogalactopyranoside is an
  inductor, but no substrate
• BCIG 5-Br, 4-Cl, 3-indolyl b-D
  galactopyranoside is a substrate, but no inductor

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Essential characteristics - expression is
permanent since IPTG is not metabolized -
intense blue color of the indigo compound -
indigo compound is extremely insoluble in water
no diffusion For analyses in solution ONPG is
used ordinarily ortho-nitrophenyl
galactopyranoside hydrolysis produces the
yellow ortho-nitrofenol (absorption at 420 nm)
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pUC2 EcoRI pUC7 symmetrical (palindromic)
polylinker EcoRI-BamHI-SalI-PstI-SalI-BamHI-Ec
oRI excision between both EcoRI sites, both
BamHI sites or both SalI sites removes a
multiple of 3 bp so that the reading frame is
unchanged. pUC8 9 asymmetrical
polylinkers (order of restriction sites is
inverted) etc. (see later at M13 vectors)
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Incorporation of bacteriophage promoters to
produce RNA probes
Labeling (radioactive) or tagging
(non-radioactive) with (one of) the dNTPs.
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Runoff transcripts transcription terminates at
the physical end of the linearized DNA molecule.
If linearization is by restriction cleavage
near the junction between insert and vector, and
if the promoter lies close enough in front of
this site of insertion, then the transcription
product will be free of vector sequences, and
hence can be directly used as probe in
hybridisation. Vectors in which
two(different) promoters are placed, as shown
above, are also named dual-promotor plasmid
vector.
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pGEM vectors
Phage promoters inside the lacZa region,
surrounding the polylinker.
ps. these vectors are also fasmids (see later)
by the presence of the f1 ori.
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LITMUS vectoren phage promoter sequences in or
flanking the MCS, and in lacZa gt production of
RNA probes (strand specific probes) gt onto 1
mg DNA up to 30-40 mg RNA can be made in vitro
gt with mutant T7 promoters choice or
orientation depending on the restriction cleavage

ps. these vectors are also fasmids (see
later) by the presence if the M13 ori.
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Bacteriophages or phages Basic
aspects "DNA travelling outside the cell " a
nucleic acid genome (DNA or RNA), covered by
protein Lytic development (cycle) - infection
process, after attachment to bacterial cell wall
(at a specific receptor) - replication inside
the cell - assembly of new particles - leaving
the cell, usually, but not always, by lysis of
the cell e.g. filamentous phages Lysogenic
development (by temperate phages) - following
infection, integration into chromosome
("prophage") (site-specific or randomly,
dependent on phage type) - remains silent during
cellular propagation and cell multiplication -
induction by external factors (e.g. DNA damage by
UV) leading to onset of lytic development -
continues as above until cell lysis e.g.
bacteriophage lambda (l) gt while developing
phage vectors besides replication, also the
survival of the phage and its route of
development should be kept into consideration.
The presence of plaques (and their appearance)
is a phenotypic trait allowing visual
identification.

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Bacteriophage vectors Filamentous phages
M13, F1, fd
Life cycle of filamentous phages
6.4 kb single-stranded circular DNA phage
particle 9 x 900 nm major coat protein gp8 minor
proteins at the tips of the filament (gp3, gp6,
gp7, gp9)
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high gene density, few intergenic regions,
replication ori() ori(-) - 500 bp near
the ori - a few bp between gene 3 and
8 F-pilus is required for infection, hence F or
F' plasmid must be in the cell J. Messing
insertion of an 800 bp lacZa fragment (HincII)
near ori in BsuI GGCC (after partial
cleavage of ds-RF) gt M13mp1 "messing
plasmids" gt M13mp vectors blue plaques
versus colorless plaques key components
gene 8 product major coat protein gene 3
product pilot protein, essential for infection
gene 2 product to start replication nicking
gene 5 product ssDNA binding protein
Initial replication is in Q mode, then switches
to rolling circle replication As soon as there
are enough gp5 product, the first strand covered
and new copies remain single-stranded. At (or
in) the membrane, gp5 is replaced by gp8, when
the new filamentous particle is secreted. The
cell is not killed. But growth of the infected
cell is retarded gt turbid plaques. (!!
filamentous phages are not lysogenic phages
!!)

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HaeIII site GGCC where Messing inserted the lacZa
fragment
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RF as a plasmid transfection (also possible
with ssDNA) insert sizes almost unlimited
(very large inserts cause shear-sensitive
phages) strand complementarity of clones with
inserts in two different orientations E.coli
D(lac-proAB) F' traD34 proAB lacIq
lacZDM15 gt selection on minimal
medium vectors random mutagenesis M13mp1 gt
M13mp2 EcoRI target enrichment by EcoRI
cleavage and gel electrophoresis polylinker
M13mp7 symmetry cloning in M13mp7 gt
recovery of inserts by excision vector pairs
M13mp8/9 ( clone turn around technique)
further derivatives (usually pairwise) with
increasing number of cleavage sites applications
where single-stranded DNA is needed or
preferred in particular for DNA
sequencing site-directed mutagenesis

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(No Transcript)
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