In 1952, Joshua Lederberg coined the term plasmid to describe any bacterial genetic element that exists
in an extrachromosomal state for at least part of its replication cycle . As this
description included bacterial viruses, the definition of what constitutes a
plasmid was subsequently refined to describe exclusively or predominantly
extrachromosomal genetic elements that replicate autonomously.
(read next)
page.
0 1
Naturally occurring plasmids vary greatly in
their physical properties, a few examples of which are shown in Table 1. They range in size from <2-kilobase pair (kbp)
plas-mids, which can be considered to be elements simply capable of
replication, to
From: Methods in Molecular Biology, Vol.
(read next)
page.
2
Although most plasmids possess a circular geometry, there are now many examples in a variety of bacteria
of plasmids that are linear (15,16).
As linear
plasmids require specialized mechanisms to replicate their ends, which circular
plasmids and chromosomes do not, linear plasmids tend to exist in bacteria that
also have linear chromosomes (17).
(read next)
page.
3
Many plasmids are phenotypically cryptic and
provide no obvious benefit to their bacterial host other than the possible
exclusion of plasmids that are incompatible with the resident plasmid (see Part 2).
(read next)
page.
4 5
Plasmids, like chromosomes, are replicated
during the bacterial cell cycle so that the new cells can each be provided with
at least one plasmid copy at cell division (41). To this end, plasmids have developed a number of
strategies to initiate DNA replication but have mostly co-opted the host
polymerization machinery (42) for subsequent stages of DNA synthesis, thereby
minimizing the amount of plasmid-encoded information required for their
replication.
(read next)
page.
6
The genetic organization of a stylized
plasmid replicon is illustrated in Fig. 2A. This replicon consists of a number of elements,
including a gene for a plasmid-specific replication initiation protein (Rep),
a series of directly repeated sequences (iterons), DnaA boxes, and an adjacent
AT-rich region.
(read next)
page.
7 8 9
The replicon of the ColEl plasmid of Escherichia coli is the basis for many gene-cloning and gene-expression
vectors that are commonly used in current molecular biology (see Parts 2 and
28). In contrast to the replication of iteron-containing plasmids, ColE1
replication proceeds without a plasmid-encoded replication initiation protein
and instead utilizes an RNA species in initiation and RNA-RNA interactions to
achieve copy number control (see Fig.
(read next)
page.
10 11
Many small (<10 kbp) plasmids of
Gram-positive Eubacteria replicate by a rolling-circle mechanism, which is
distinct from the replication of iteron-containing or ColE1-like plasmids (see Fig. 3) (47).
(read next)
page.
12 13
DNA replication produces precise plasmid
copies, but plasmids must also ensure that they are distributed to both
daughter cells during bacterial cell division. If the
Fig. 3. Replication of rolling-circle
plasmids.
(read next)
page.
14 15
Following plasmid replication, active partitioning
systems position the plasmids appropriately within the cell such that at cell
division, each of the new cells acquires at least one copy of the plasmid (see Fig.
(read next)
page.
16
Many laboratory strains of E. coli have been mutated to be deficient in homologous
recombination. This reduces the frequency with which genes cloned in multicopy
plas-mids undergo rearrangements in these strains.
(read next)
page.
17 18 19
An additional mechanism which plasmids use
to favor their maintenance in bacterial populations involves the killing or
growth impairment of cells that fail to acquire a copy of the plasmid. This has
variously been referred to as postsegregational cell killing, plasmid
addiction, or toxin-antitoxin systems (57-60).
(read next)
page.
20 21
Certain bacterial species can achieve a
state of natural competence for the uptake of naked plasmid DNA
(transformation) (62), or can acquire DNA that has been packaged into a
bacteriophage head and is injected into the host (transduction) (63).
(read next)
page.
22 23 24
Whole genome and plasmid-specific sequencing
projects have recently begun to provide fascinating glimpses into the genetic
organization and evolution of plasmids. These studies have revealed that
plasmids, particularly large plasmids, are commonly constructed in a modular
fashion by the recombination activities of transposons, insertion sequences,
bacteriophages, and smaller plasmids (72).
(read next)
page.
25 26 27
Since the construction of the first
generation of general cloning vectors in the early 1970s, the number of
plasmids created has increased to an almost countless number. Thus, a critical
decision facing today's investigator is that of which plasmid to use in a
particular project?
(read next)
page.
28
For projects in which it is desired that a
particular piece of DNA be cloned, one consideration is the size of the insert
DNA. Most general cloning plasmids can carry a DNA insert up to around 15 kb in
size.
(read next)
page.
29
Cosmids are conventional vectors that
contain a small region of bacteriophage X DNA containing the cohesive end site
(cos). This contains all of the cis-acting elements for packaging of viral DNA into X
particles.
(read next)
page.
30
The bacteriophage X genome comprises 48,502 bp.
On entering the host cell, the phage adopts one of two life cycles: lytic
growth or lysogeny. In lytic growth, approx 100 new virions are synthesized and
packaged before lysing the host cell, releasing the progeny phage to infect new
hosts.
(read next)
page.
31 32
Bacterial artificial chromosomes (BACs) are
circular DNA molecules. They contain a replicon that is based on the F factor
comprising oriS and repE encoding an ATP-driven helicase along with parA, parB, and parC to facilitate accurate partitioning (see Part 1).
(read next)
page.
33
Different cloning vectors are maintained at
different copy numbers, dependent on the replicon of the plasmid (see Part 1).
In a majority of cases in which a piece of DNA is cloned for maintenance and
amplification for subsequent manipulation, the greater the yield of recombinant
plasmid from E.
(read next)
page.
34 35
Incompatibility refers to the fact that
different plasmids are sometimes unable to coexist in the same cell. This
occurs if the two different plasmids share functions required for replication
and/or partitioning into daughter cells.
(read next)
page.
36
Introduction of plasmids in to E. coli cells is an inefficient process. Thus, a method of
selecting those cells that have received a plasmid is required. Furthermore,
cells that do not contain a plasmid are at a growth advantage over those that
do and, thus, have to replicate both the chromosome and additional plasmid DNA.
(read next)
page.
37 38 39
This drug inhibits the bacterial transpeptidase
involved in peptidoglycan biosynthesis and thus inhibits cell wall
biosynthesis (14). As such, ampicillin inhibits log-phase bacteria but not
those in a stationary phase.
(read next)
page.
40
A member of the aminoglycoside family of
antibiotics, kanamycin was first isolated from Streptomyces kanamyceticus in Japan in 1957. This polycation is taken into the
bacterial cell through outer-membrane pores but crosses the cytoplasmic membrane
in an energy-dependent process utilizing the membrane potential.
(read next)
page.
41
First isolated from a soil actinomycete in
1947, chloramphenicol was widely used as a broad-spectrum antibiotic although
its clinical use has been curtailed because of drug-induced bone-marrow
toxicity and the emergence of bacterial chloramphenicol resistance.
(read next)
page.
42
Originally isolated from Streptomyces aureofaciens in 1948, there are now many tetracycline derivatives
available. They bind to a single site on the 30S ribosomal subunit to block the
attachment of aminoacyl tRNA to the acceptor site and thus inhibit protein
synthesis (19).
(read next)
page.
43
The cloning of DNA into a vector usually
involves ligation of the insert DNA fragment to vector DNA that has been cut
with a restriction endonuclease. This is facilitated by the insert and vector
DNA fragments having compatible cohesive ends.
(read next)
page.
44 45
Some projects will involve specific
downstream applications that will require specialized plasmid functions that
are only present on some plasmids. For example, both the pUC and pBluescript
series of vectors are high-copy-number, ampicillin-resis-tance-conferring
plasmids that contain MCSs that facilitate the use of a wide range of
restriction endonucleases in the cloning step.
(read next)
page.
46 47
When choosing a cloning vector for use in a
cloning project, the investigator is faced with an enormous choice. However,
the application of a small number of criteria can quickly guide the selection
of a suitable vector.
(read next)
page.
48
To successfully perform molecular genetic
techniques it is essential to have a full understanding of the properties of
the various Escherichia
coli host strains
commonly used for the propagation and manipulation of recombinant DNA.
(read next)
page.
49 50
A genotype indicates the genetic state of
the DNA in an organism. It is associated with an observed behavior called the
phenotype. Genotypes of E. coli strains are described in accordance with a standard
nomenclature proposed by Demerec et al.
(read next)
page.
51
The genotypes and features of a
representative selection of popular host strains used for general recombinant
DNA cloning procedures are listed in Table 2. An extended listing of available strain genotypes can
be found in ref.
(read next)
page.
52
Many laboratory E. coli strains carry mutations that reduce their viability in
the wild and preclude survival in the intestinal tract (6). These often confer auxtrophy, that is, they disable the
cell's ability to synthesize a critical metabolite, which, therefore, must be
supplied in the medium.
(read next)
page.
53
Some vectors contain nonsense mutations in
essential genes as a means of preventing spread to natural bacterial
populations. Nonsense mutations are chain-termination codons; they are termed
amber (UAG) or ochre (UAA) mutations (5).
(read next)
page.
54
Some E. coli strains carry an F episome or fertility factor, which
can be found in several different forms (7). It may be carried as a double-stranded single-copy
circular extrachromosomal plasmid, designated F+, or if it harbors additional
genes, F'.
(read next)
page.
55
Restriction-modification systems play a role
in preventing genetic exchange between groups of bacteria by enabling the host
to recognize and destroy foreign DNA. An archetypal system consists of a DNA
methylase and its cognate restriction endonu-clease.
(read next)
page.
56
Derivatives of E. coli K-12 normally contain three site-specific DNA
methylases: Dam, Dcm and EcoK. DNA adenine methylase, encoded by dam, methylates
adenine residues in the sequence GATC (9,10).
(read next)
page.
57 58
The E. coli K-12 EcoK methylase modifies the indicated adenine residues of
the target sequence A(mA)CN6GTGC, and its complement GC(mA)CN6GTT
(8,16). The cognate endonuclease will cleave DNA that is
unmodified at this sequence.
(read next)
page.
59
E. coli K-12 also contains several methylation-dependent
restriction systems, namely McrA, McrBC, and Mrr. The methylcytosine
restricting endonucleases, McrA and McrBC, cleave methylcytosines in the
sequences CG and (A/C)G, respectively (1821).
(read next)
page.
60
Following successful transformation of a
plasmid vector into E. coli,
host recombination
systems can catalyze rearrangement of the recombinant molecule. This is a particular
problem when the cloned DNA contains direct or inverted repeats and can result
in duplications, inversions, or deletions.
(read next)
page.
61 62 63
Bacteriophage X is injected into the E. coli host as a linear molecule that rapidly circularizes
and, during the early phase of infection, replicates by a bidirectional 9-type
mechanism, yielding monomeric circles.
(read next)
page.
64 65
Many current molecular biology techniques
rely on the pioneering studies of the lac operon
by Jacob and Monod in the 1960s (46). The lac operon consists of three genes: lacZYA, encoding p-galactosidase, which cleaves lactose to
glucose and galac-tose, a permease, and a transacetylase.
(read next)
page.
66 67
The frequency of spontaneous mutation in E. coli may be increased by three to four orders of magnitude
by mutations in mutD, which encodes the 3'—5' exonuclease sub-unit of the DNA
polymerase III holoenzyme (50,51).
(read next)
page.
68 69
E. coli is a popular host for the overexpression of recombinant
proteins (see Parts 28 and 29). There are a number of factors that
can influence protein yields and careful strain choice can greatly improve the
chance of successful expression.
(read next)
page.
70
E. coli expression vectors utilize highly active inducible
promoters and the correct host strain must be used to ensure proper tight
regulation (53). Many common vectors
Table 4
Properties of E.
(read next)
page.
71 72
Host proteases can interfere with the
isolation of intact recombinant proteins; degradation may be avoided by the
use of protease-deficient hosts. In E. coli, lon encodes a major ATP-dependent protease and strains that
contain deletions of this gene greatly improve the yield of many recombinant
proteins (54,55).
(read next)
page.
73 74
The frequency with which amino acid codons
are utilized varies between organisms and is reflected by the abundance of the
cognate tRNA species. This codon bias can have a significant impact on heterologous
protein expression, so that genes that contain a high proportion of rare codons
are poorly expressed (61,62).
(read next)
page.
75
Overproduction of heterologous proteins in E. coli often results in misfolding and segregation into
insoluble inclusion bodies. The cytoplasmic chaperones, DnaK-DnaJ and
GroES-GroEL, assist proper folding in wild-type E.
(read next)
page.
76
Since the first mutants of E. coli K-12 were isolated in the 1940s, laboratory strains
have been heavily mutagenized by treatment with X-rays, ultraviolet
irradiation, and nitrogen mustard. Thus, they may carry unidentified mutations
and it can be useful to try more than one strain background if experiments are
unsuccessful.
(read next)
page.
77
Transformation is defined as the transfer of
genetic information into a recipient bacterium using naked DNA, without any requirement
for contact with a donor bacterium. The ability to transform or accept
exogenous DNA is generally referred to as competence, although the term has
been so widely used in different systems that it is difficult to generate an
all-inclusive definition for competence.
(read next)
page.
78 79
Preparation of
Competent Cells
Classical
Calcium Chloride Method
1. Host
bacterial strain (see Note 1).
2. Luria-Bertani
(LB) broth: 5 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl.
(read next)
page.
80 81
- Preparation
of Competent Cells
- Classical Calcium Chloride Method (2,3)
This method was the first generally
applicable method for transformation of E. coli with plasmid DNA with typical yields of 1 x 107
transformants per microgram of DNA and is still in wide use.
(read next)
page.
82 83 84 85
1. For
factors affecting the choice of host strain, see Part 3.
2. Place
solution on ice early in the growth of the bacteria to ensure that it is
thoroughly chilled before use.
3. If the
cells are to be stored at -70°C, use ESB buffer rather than TFB.
(read next)
page.
86 87 88
Electroporation, originally developed as a
method to introduce DNA into eukary-otic cells (7), has
subsequently been extensively used for bacterial transformation (2,3). This procedure is an effective method for the transfer
of DNA to a wide range of Gram-negative bacteria, such as Escherichia coli, and reports indicate that 109
electro-transformants per microgram of DNA can be achieved in this species (4,5).
(read next)
page.
89 90 91 92
1. E. coli strain (see Note 2).
2. Luria-Bertani
(LB) broth medium: 10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L of sodium
chloride. Adjust to pH 7.0 by addition of 5 N NaOH;
autoclave.
(read next)
page.
93
Preparation of
E. coli Electrocompetent Cells
1. Streak a
suitable E. coli strain onto an LB agar plate for single colonies and
incubate at 37°C overnight.
2. Inoculate
50 mL of LB medium with a single colony of freshly grown E.
(read next)
page.
94 95 96
1. To
electroportate ElectroMAX™DH5a-E E. coli cells using the Bio-Rad Gene Pulser unit, the following
conditions are used to yield approx 1.0 x 1010 transformants per
microgram pUC plasmid DNA: 1.8
(read next)
page.
97 98 99
Bacterial conjugation is defined as
contact-dependent transmission of genetic information from a donor bacterium to
a recipient cell (7). Transfer of DNA by conjugation is often termed lateral or horizontal
gene transfer, as opposed to
vertical transfer by which genetic information is transferred from mother to
daughter cells.
(read next)
page.
100 101 102 103 104
1. Luria-Bertani
(LB) broth medium: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium
chloride. Adjust to pH 7.0 by addition of 5 N NaOH and
autoclave.
2. Antibiotics
for selection of transconjugants.
(read next)
page.
105
1. Dilute
overnight cultures of the donor and recipient strains 1 in 50 in fresh LB
broth. Incubate at 37°C with vigorous shaking until an OD600 of 0.6
- 0.8 is reached.
2. Mix
different ratios of the donor and recipient strains in a sterile universal.
(read next)
page.
106
1. Different
ratios of donor to recipient strains are used to optimize the conjugation procedure.
It may also be necessary to increase the cell biomass of the bacterial
cultures. Optimization is particularly important if the recipient strain is
not E.
(read next)
page.
107 108
Cosmids are cloning vectors that were
developed to enable large fragments of DNA to be cloned and maintained (1-3). Cosmid vectors allow the cloning of fragments up to 45 kilobases
(kb) and are commonly used in genomic library construction.
(read next)
page.
109 110 111 112
Ligation
Reaction
1. Prepared
(restriction digested and phosphatase treated) vector DNA (e.g., SuperCos I
[Stratagene]). Store at -20°C.
2. Prepared
(restriction digested and phosphatase treated) genomic DNA.
(read next)
page.
113
Ligation
Reaction (1,3,6,10-13)
1. Set up the following ligation reaction in
a microcentrifuge tube:
1.5-3.0 prepared genomic DNA (32-45 kb
in length) (see Note 4). 1.0-3.0 prepared vector DNA.
(read next)
page.
114 115 116
1. One of
the most important things to consider when constructing a cosmid library is the
efficiency of the packaging extracts. It is extremely important that the
packaging extracts are not allowed to thaw before use.
(read next)
page.
117 118 119
Purification of plasmid DNA from Escherichia coli using alkaline lysis (1,2) is based on the differential denaturation of
chromosomal and plasmid DNA in order to separate the two. Bacteria are lysed
with a solution containing sodium dodecyl sulfate (SDS) and sodium hydroxide.
(read next)
page.
120 121
Growth of E. coli
1. Luria-Bertani
(LB) medium: 5 g/L yeast extract, 5 g/L NaCl, 10 g/L tryptone.
2. Appropriate
antibiotics.
Plasmid
Isolation
1. STE (sucrose/Tris/EDTA)
solution: 8% (w/v) sucrose, 50 mM Tris-HCl (pH 8.0
(read next)
page.
122
Growth of E. coli
1. Inoculate
3 mL of sterile LB medium containing the appropriate antibiotic with a single
bacterial colony.
2. Grow
with shaking at 37°C overnight.
Plasmid
Isolation
1.
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page.
123 124 125
1. The
original protocol asks for 0.2 N NaOH. However, if the isolated plasmid DNA is to be
used in sequencing reactions, reducing the NaOH concentration to 0.1 N is recommended.
This reduces the amount of nicked and denatured DNA (see Note 7) without a significant impact on DNA yield.
(read next)
page.
126 127 128
The boiling lysis procedure (1) is quick to perform and, therefore, especially suitable
for screening large numbers of small-volume Escherichia coli cultures. It is described with different adaptations in
a variety of protocol books (2,3).
(read next)
page.
129 130
Growth of E. coli
1. Luria-Bertani
(LB) medium: 5 g/L yeast extract, 5 g/L NaCl, 10 g/L tryptone. Autoclaved.
2. Appropriate
antibiotics.
Plasmid
Isolation
1. STE solution: 8% (w/v) sucrose, 50 mM
Tris-HCl (pH 8.0
(read next)
page.
131
Growth of E. coli
1. Inoculate
3 mL of sterile LB medium containing the appropriate antibiotic with a single
bacterial colony.
2. Grow
with shaking at 37°C overnight.
Plasmid
Isolation
1.
(read next)
page.
132 133 134 135 136
The isolation of plasmid DNA from bacteria
is a crucial technique in molecular biology and is an essential step in many
procedures such as cloning, DNA sequencing, transfection, and gene therapy.
(read next)
page.
137 138 139
1. Silica
oxide (Sigma): Dissolve in 250 mL of water, at 50 mg/mL, for 30 min. Remove the
fines by suction and reconstitute the original volume. Add 150 of 37% HCl and
autoclave.
2. P1: 50 mM Tris-HCl,
10 mM EDTA (pH 8.0
(read next)
page.
140
1. Harvest 1.5-2
mL of overnight cultures of Escherichia
coli clones of
interest in Eppendorf tubes by centrifugation at 1000g for 5 min
and completely remove the supernatant (see Notes 2-4).
2.
(read next)
page.
141 142
1. To
facilitate pipetting, acetone should be stored at -20°C.
2. This
method can be scaled up for larger cultures; recommended volumes of solutions
to use in the different formats are given in Table 1.
(read next)
page.
143 144
Plasmid extraction is typically performed to
produce template DNA for a desired molecular biological reaction, or set of
reactions, such as restriction endonuclease digestion (see Part 20),
DNA sequencing (see Part 22), in vitro mutagenesis (see Parts 23-26),
transformation (see Parts 4 and 5), transfection, or probe generation.
(read next)
page.
145 146 147 148 149 150 151
96-Well
Miniprep of Plasmid DNA
1. Circlegrow
(Anachem, UK).
2. Ampicillin,
or other antibiotic as appropriate.
3. Deep-well
96-well plates (Beckman).
4. Plate
sealer (Costar, Corning).
(read next)
page.
152 153
Plasmid
Preparation in 96-Well Format
1. Fill
each well of a 96-well-deep well plate with 1 mL of Circlegrow containing the
appropriate antibiotic (typically ampicillin at a final concentration of 100 ^g/mL)
(see Notes 1, 3, and 4).
(read next)
page.
154 155
1. Fill
each well of a 96-well deep-well plate (see Notes 1,
3, and 4) with 1.25
mL of 2X TY containing the appropriate antibiotic (typically 25 yg/mL of
kanamycin for PACs and cosmids or 12.5 yg/mL of
chloramphenicol for BACs).
(read next)
page.
156 157
1. Fill
each well of a 96-well deep-well plate (see Notes 1,
3, and 4) with 1.25
mL of 2X TY that has been seeded with a 1% (v/v) inoculum of an overnight
culture of an appropriate M13 host strain.
(read next)
page.
158 159
1. Deep-well
plates can be filled manually using a reservoir-based repeat pipettor such as
an Eppendorf multipipet or by using a 1-mL-capacity multichannel pipet. For
filling large numbers of boxes, a 96-well dispensing unit such as a Q-fill (25) is
recommended.
(read next)
page.
160 161 162
Cosmid and bacterial artificial chromosome
(BAC) systems have been developed for the cloning of large DNA inserts
averaging 40 kb and 130 kb (range: 90-300 kb), respectively. The resulting
clones are more stable than yeast artificial chromosomes (YACs) and rarely
chimeric, which makes them excellent tools for the generation of contiguous physical
maps.
(read next)
page.
163 164 165
1. Luria-Bertani
(LB) medium: 10 g Bacto tryptone, 5 g Bacto yeast extract, 10 g NaCl; make up
to 1 L with double-distilled water (ddH2O). Sterilize by
autoclaving.
2. Terrific
Broth (TB): 12 g Bacto tryptone, 24 g Bacto yeast extract, 10 mL of 40% (v/v)
sterile glycerol, 17 mL of 1 M KH2PO4, 72 mL of 1 M K2HPO4;
(read next)
page.
166
(see Notes 1 and 2)
1. Prepare
a 100- to 150-mL culture of the cosmid or BAC clone to be purified in LB or TB
media containing the appropriate antibiotic (50 yg/mL
kanamycin or 100 yg/mL ampicillin for cosmids and 12.5
(read next)
page.
167 168 169
1. This
protocol can be adapted for minipreparation of DNA (3).
2. Cosmids
and BACs can also be prepared using the CONCERT™ High Purity Plasmid
Purification System from Invitrogen. However, it is necessary to modify their
midiprep protocol as follows.
(read next)
page.
170 171
Single-stranded DNA (ssDNA) is the optimal
template for most polymerase-based molecular-biology applications, including
DNA sequencing and site-directed mutagenesis. Phagemids are chimeric vectors,
derived from the ssDNA bacteriophages M13, fd, or f1, that normally replicate
as plasmids in bacterial hosts (1) (see Part 2).
(read next)
page.
172 173
Determination
of Helper Bacteriophage Titer
1. Luria-Bertani
(LB) broth: 5 g tryptone, 10 g yeast extract, 5 g NaCl. Bring to 1 L with water
and autoclave.
2. LB agar:
Add 15 g Bacto agar to 1 L of LB broth and autoclave.
(read next)
page.
174
Determination
of Helper Bacteriophage Titer
1. Prepare
fifteen 5-mL aliquots of semisolid LB top agar in glass tubes and place at 42°C
to keep the agar molten.
2. Add 108
colony-forming units of a susceptible bacterial strain to 0.5
(read next)
page.
175 176 177
1. It is
important to use good bacteriological techniques. Start with single-colony
inoculae and carefully monitor the growth of the bacterial cultures.
2. The titer
of the helper phage is important and should be determined in plaque assays as
described in Subheading 3.1
(read next)
page.
178
Manipulation and analysis of DNA sequences
is often a complex task involving many steps, each of which must be carefully
planned and executed. To facilitate this process, the number of steps should be
minimized and each step analyzed to ensure that it has been completed
successfully.
(read next)
page.
179 180 181
Background
As an example, an actual cloning project
will be described. In this project, the coding sequence of a Drosophila heat-shock gene (hsp26) was cloned into a vector downstream of a regulated
promoter.
(read next)
page.
182 183
Identifying
Open Reading Frames
1. To
characterize pRmHa3, coding regions were identified by highlighting open
reading frames (ORFs) using Construct
> Features > Find Open Reading Frames (see Fig.
(read next)
page.
184 185 186
Creating a New
Generation
1. The
source of the hsp26 coding sequence was the plasmid pJBl (see Fig.
5A). In order to
isolate the segment containing the hsp26 coding sequence, pJ1B was digested with EcoRI and BamHI and the smaller of the two fragments generated was
isolated using gel electrophoresis and gel extraction.
(read next)
page.
187 188 189 190 191 192
1. GCK's
Deluxe Importing feature (File >
Deluxe Import > Search GenBank) allows for a straightforward importing of GenBank (or
EMBL) sequence files directly from the corresponding websites.
(read next)
page.
193
1. Another useful feature of GCK, although
not shown here, is the ability to create partial digests. For example, it is
possible to specify that during a digest only five of six sites are cut. The
resulting digest pattern will show complete digest fragments as solid black
lines (bands) and partial digest fragments as dotted blue lines.
(read next)
page.
194 195
A fundamental step in molecular biology is
the cloning of a DNA fragment insert into a plasmid vector. This allows the
cloned fragment to be replicated upon transformation of the recombinant
molecule into a bacterial cell (see Parts 4 and 5) so that the DNA of interest can be
investigated further.
(read next)
page.
196 197
The first step in cloning a DNA insert into
a plasmid vector is cutting both vector and insert DNA with the appropriate
restriction enzyme(s) to generate compatible ends. This may be a simple single
digestion or a double digestion with two enzymes in the case of directional
cloning.
(read next)
page.
198 199
If the insert DNA does not contain
convenient restriction sites, it is possible to generate a site at the desired
position by amplifying the insert using the PCR primers designed with the
restriction site.
(read next)
page.
200
In cloning experiments where compatible ends
are not available, it may be necessary to convert a 5' or 3' overhang to a
blunt end (see Fig. 1B). Both bacteriophage T4 DNA polymerase and Escherichia coli DNA polymerase I large (Klenow) fragment have 5'—3' polymerase
activity and can be used to fill in 5' overhangs.
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Alkaline phosphatases are commonly used in
cloning experiments to dephosphory-late the 5' ends of vector DNA. This
prevents self-ligation of the vector, as the enzyme used to ligate the DNA
molecules requires a 5'-phosphate group on one of the DNA substrates (see Fig.
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page.
202
Phosphorylation of insert DNA that lacks
terminal 5' phosphates, such as PCR products and fragments with synthetic
linkers, may be required in preparation for ligation. If the product is to be
cloned into a nonphosphorylated vector, it is vital that phosphate groups are
added to the insert.
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203
The final step in cloning is the joining of
the linear DNA fragments together, referred to as ligation. This involves
creating a phosphodiester bond between the 3'-hydroxyl group of one DNA
fragment and the 5'-phosphate group of another and is equivalent to repairing
nicks in a duplex strand.
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Restriction
Digestion
1. Appropriate
restriction enzyme supplied with buffer; store at -20°C.
2. 1 mg/mL
bovine serum albumin (BSA), acetylated.
3. 0.5 M EDTA (pH
8.0).
4.
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205 206
Restriction
Digestion
Complete
Digests
1. Add the
following to a 1.5-mL Eppendorf tube on ice (see Note 3):
DNA 0.1-1 yg (see Note 4) x yL
Restriction enzyme (see Notes
5-7)1 yL
Restriction enzyme 10X reaction buffer (see Note 8)2 yL
BSA 1 mg/mL 2 yL
Sterile double-distilled water to a final
volume of 20 yL(see Note 9)
2.
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1. ATP
should be present at a concentration of at least 1 yM.
2. The
buffer is usually provided or prepared by the manufacturer as a 10X
concentrate, which, on dilution, yields an ATP concentration of approx 0.2
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A common step in cloning experiments is the
purification of DNA fragments prior to ligation. Often, both the insert and
vector DNA fragments will be derived from restriction endonuclease digests and,
thus, will be mixed with enzymes, salts, and possibly other DNA fragments that
may inhibit the ensuing ligation reaction.
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page.
225
2.1. Low-Melt
Agarose Protocol
1. Low-melting-point
agarose.
2. 5X TBE
buffer: 54 g Tris base, 27.5 g boric acid, 20 mL of 0.5 M EDTA (pH
8); make up to 1 L with distilled water. Dilute the stock to give a 1X working
solution immediately prior to use.
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226
3.1. Low-Melt
Agarose Protocol
This protocol makes use of low-melt agarose.
In its simplest form, this protocol is more an avoidance of extraction rather
than an extraction per se. It can be modified to increase the purity of the DNA
sample (see Subheading 3.1
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227 228 229
1. If TBE
electrophoresis buffer is inhibitory to the downstream application and the DNA
is to be used without further purification, it is possible to use a different
buffer system for gel electrophoresis.
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page.
230 231
1.1. Overview
of PCR
Since it was described in 1988 (/), the
polymerase chain reaction (PCR) has been a valuable tool for molecular
biologists. PCR allows researchers to produce a large quantity of a desired
DNA fragment while requiring only a small amount of template.
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page.
232 233 234 235 236 237 238 239 240
2.1. Preparation
of an Xcml-Based T-vector
2.1.1. Construction
of Custom T-Vector
1. Sterile
distilled water.
2. 1 yg/yL
Oligonucleotide #1 in water: 5'-GATCCAAGCTTCCCATGGCGCCATGTCAT GAGTGGCTGCA-3'.
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page.
241 242
3.1. Preparation
of an Xcml-Based T-Vector
3.1.1. Construction
of Custom T-Vector
3.1.1.1. Preparation
of Oligonucleotides
1. Combine 25
yL oligonucleotide #1 and 25 yL
oligonucleotide #2 in a microcentrifuge tube.
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1. In the
past, researchers have had difficulty with XcmI
performance and this hampered preparation of T-vectors. However, higher-quality
enzyme is now available and this problem is not as prevalent as it once was.
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Lambda (k) bacteriophages are viruses that
specifically infect bacteria. The genome of k-phage is a double-stranded DNA
molecule approx 50 kb in length (7). In bacterial cells, k-phage employs one of two pathways
of replication: lytic or lysogenic.
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2.1. Preparation
of Genomic DNA for Cloning
2.1.1. Purification
of Genomic DNA
We suggest the use of the Wizard Genomic DNA
Purification Kit (Promega); items 1-3
are components
of that kit.
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3.1.
Preparation of Genomic DNA for Cloning
3.1.1. Purification
of Genomic DNA
1. Mince 150
mg of tissue in 40 yL/mg of ice-cold nuclei lysis buffer. Homogenize on ice
using 10-15 strokes with a Teflon pestle.
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1. A number
of similar ready-to-use k-vectors are commercially available for genomic
library construction. For example, LambdaGEM-11 BamHI arm
is a similar vector and is currently available (Promega).
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page.
270 271 272 273
Construction of recombinant plasmid DNA is
one of the cornerstones of molecular biology. The ability to clone DNA in a
plasmid vector opens doors to downstream applications such as amplification of
DNA, expression of desired genes, and construction of DNA libraries.
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2.1. Blue-White
Selection
1. Luria-Bertani
(LB) agar plates: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 15 g agar. Add
water to 1 L and autoclave to sterilize. Cool to approx 50°C, add antibiotics
as appropriate, and pour approx 20 mL into each Petri dish.
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page.
279 280
3.1. Blue-White
Selection
1. Transform
E. coli with the ligation reaction using methods described in Parts
4 and 5.
2. Prepare
plates by adding 40 yL of 20 mg/mL X-gal and 50 yL of 0.1
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1. Instead of spreading IPTG and X-gal on to
LB agar plates, they can be added to the agar mixture before plates are poured
(final concentration: 6 mM IPTG and 0.3 mg/mL X-galin LB agar). However, this
approach uses more IPTG and X-gal and these plates have a fairly short shelf
life.
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285 286
A key step in the construction of
recombinant plasmids is the verification of the successful cloning of insert
DNA into the vector. A number of commonly used plas-mids facilitate phenotypic
selection and/or screening methods for rapid identification of
insert-containing clones.
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The choice of appropriate enzyme(s) for the
restriction analysis of the clone will depend on the plasmid and insert
involved. Several criteria may influence this decision. For example, the
resulting DNA fragments need to be within a size range detectable on a gel (see Note 1) and the fragments of interest must be easily distinguishable
from each other.
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2.1.
Restriction Enzyme Digestion
1. Predicted
restriction map of the plasmid clone. Prepare a map for a clone with the insert
in each of the two possible orientations if applicable.
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3.1. Restriction
Enzyme Digestion
1. Thaw all
solutions, except the enzyme, and keep on ice.
2. Using a
final volume for the digest of 20 yL (or up
to 50 yL if the DNA is dilute), add the following into a
sterile Eppendorf tube:
1/10 volume reaction buffer (see Note 9).
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294 295
1. A DNA
fragment of a size between 100 and 10,000 base pairs is ideal. DNA fragments
that are larger or smaller than this will migrate in the gel, but may blur or
fail to resolve (1,4,5). If
trying to resolve and analyze fragments larger than 5 kb, a gel that is longer
than a mini-gel is required.
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A recombinant DNA library typically
represents part or all of an organism's genomic DNA or mRNA (represented as
cDNA) cloned into vectors and stored as a collection of thousands of
transformants.
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302
1.1.1. Phenotypic
Screening
In a small number of cases, a cloned
fragment of DNA will possess an intact gene that encodes a protein of
discernable function. Some examples are genes encoding pigments, secreted
enzymes, or assayable metabolic functions.
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2.1. Preparing
the Membrane
1. Recombinant
library stored in multiwell plates.
2. LB agar:
10 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, and 15 g/L bacto-agar.
Sterilize by autoclaving.
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313 314
3.1. Preparing
the Membrane
1. Remove a
multiwell plate containing the library from the freezer and allow the bacterial
suspensions to thaw on ice.
2. Label
the edge of a nitrocellulose membrane with the specific information about the
library (plate number, date, etc.)
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The most widespread method used for DNA
sequencing today is the Sanger dideoxy method that was first described in 1977 (7). This
method takes advantage of the requirement for a free 3' hydroxyl group to form
the necessary phosphodiester bridge between two nucleotides during DNA
polymerization.
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2.1. Sequencing
Reaction Setup
1. Purified
plasmid (100-500ng/yL), polymerase chain reaction (PCR) product (10 ng per 100
bp), or bacterial artificial chromosome (BAC) clone (200-600 ng/yL) (see Note 1).
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3.1. Sequencing
Reaction Setup (see Note 8)
3.1.1. Reaction Setup
for Slab Gel Sequencing of Plasmids and PCR Products
1. Add 1-5 yL of
plasmid DNA or 10 ng per 100 bp PCR template DNA to a sterile, thin-wall PCR
tube or a 96-well PCR plate.
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1. Template
DNA must be of extremely high quality. When using the ABI dye chemistries,
purify plasmid templates using Qiagen mini-spin kits (www.qiagen.com)
or Promega Wizard preps (www.promega.c
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Site-directed mutagenesis (SDM) is used to
introduce a defined mutation into target DNA of known sequence to study, for
example, gene expression or protein structure-function relationship. A number
of polymerase chain reaction (PCR)-based mutagenesis methods have been developed
(7).
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344 345 346
1. DNA template
and plasmid carrying the gene sequence to be mutated.
2. Oligonucleotide
primers: Two external primers (forward and reverse) and one internal mutagenic
primer.
3. 5 U/yL Pfu DNA
polymerase and 10X reaction buffer (Stratagene, La Jolla, CA).
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3.1. Primer
Design
3.1.1. Mutagenic
Primer
We design primers that are 22-24 bp in
length. This gives sufficient length for incorporating the required base-pair
change and to give the desired annealing temperature (Tm) >
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1. Template
DNA should be kept at a low concentration (e.g., around 1 ng). Excess template
leads to high levels of wild-type sequence being carried over into the
second-round PCR, which results in a high level of wild-type sequence in the
second-round PCR products.
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352 353
Site-directed mutagenesis has revolutionized
the study of protein structure and function by enabling the controlled and
systematic production of mutant proteins. Early methods of site-directed
mutagenesis involved the use of a mutated oligonucleotide primer to prime
synthesis of a target single-stranded DNA template.
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page.
354 355 356
Enzymatic inverse PCR using Type IIS
restriction endonucleases (EIPCR-IIS) is a significant improvement over the
classical method (4). In this technique, the 5' termini of both primers
contain a unique Type IIS restriction site, such as SapI.
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page.
357
The author's laboratory has adapted the
original EIPCR protocol for the use of class II restriction enzymes (5), thereby
extending the versatility of the technique. The principle difference from
EIPCR-IIS is that in stage 1 of this process, a unique Type II enzyme
recognition site is artificially introduced into the construct.
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3.1. EIPCR-IIS
The EIPCR-IIS protocol is outlined in Fig. 2.
3.1.1. Primer Design
Careful primer design is crucial for the
success of any DNA amplification experiment and is particularly critical when
designing primers for site-specific mutagen-esis.
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1. Magnesium
chloride is required for the activity of the DNA polymerase and is typically
used at 1.5 mM final concentration, although variation of Mg2+
levels between 1.0 and 2.5 mM MgCl2 can increase the specificity of the
amplification reaction (7).
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DNA fragments cloned into plasmids are
frequently greater than 500 base pairs in length and thus may be too long to
sequence from a single primer-binding site in the vector. An efficient way to
sequence such large DNA inserts is to generate a nested set of deletions in the
target DNA, effectively moving the priming site closer to the sequence of
interest.
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2.1. Restriction
Enzyme Digestion
1. CsCl/ethidium
bromide-purified plasmid DNA (see Note 1).
2. Restriction
enzymes and corresponding 10X buffers suitable for generating a 3' recessed
terminus or blunt end, and a four nucleotide 3' overhang (see Notes 2-4).
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381 382
3.1. Restriction
Enzyme Digestion
1. Digest 10
yg of the plasmid DNA with the restriction enzyme that
generates the 3' recessed terminus or blunt end according to the manufacturer's
instructions (this enzyme site must lie closest to the target DNA).
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1. The
generation of ordered sets of deletions by this method relies on the uniform
digestion rate of exonuclease III. However, the enzyme also digests from nicks
in double-stranded DNA molecules, creating single-stranded gaps.
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Transposons are mobile genetic elements with
the capacity to "jump" to new target DNA. Although first discovered
in Zea mays by McClintock (7), they are
present in DNA genomes of species from all kingdoms.
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2.1. Tn7-Based
In Vitro Mutagenesis of Plasmids and Cosmids
1. Tn7 transprimer
kit: Genome Priming System, GPS-1 (New England Biolabs). This kit contains the
basic transprimer plasmid (with either chloramphenicol- or kanamycin-resistance
markers), purified Tn proteins (A, B, C*), Tn reaction buffers, and Tn7-specific
sequencing primers.
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400 401
3.1. Tn7-Based
In Vitro Mutagenesis of Plasmids and Cosmids
1. Thaw the
contents of the Tn7 GPS transprimer kit and place on wet ice.
2. Prepare
the following reaction mixture in a 1.5
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In this part, we describe the use of plasmid
vectors in transcription and translation systems in vitro to investigate
aspects of the biology of the gene and the protein for which it codes. An in
vitro, or cell-free, assay reproduces a relatively complex physiological
process by mixing the essential purified components of the system under controlled
conditions outside of the cell.
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2.1. In Vitro
Transcription from Phage Promoters (see Note 1)
1. Linear
template DNA (0.2-1 yg/yL) (see Notes 2-4).
2. 5X
transcription buffer: 200 mM Tris-HCl (pH 7.9), 30 mM MgCl2,
10 mM spermidine, 50 mM NaCl.
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3.1. In Vitro
Transcription from Phage Promoters
1. Prepare the reaction mixture at room
temperature (see Note 8), as follows:
5X Transcription buffer
4
yL
100 mM DTT
2
yL
Ribonuclease inhibitor
20-40
U
2.5
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1. All
reagents, except items 1, 8, and 11, can be purchased either as separate items or as a
Riboprobe kit from Promega. Store at -20°C.
2. The gene
of interest must be cloned under the control of a strong promoter such as T3,
T7, SP6, or an appropriate E.
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Escherichia coli is the most commonly used and best characterized
organism for overexpressing foreign and nonforeign proteins. The use of E. coli confers several immediate advantages to the user: rapid
and high-level expression as a result of the speed of cell growth to high
density;
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429 430
2.1. Transcription
Versus Translation Vectors
There are two types of expression vector:
transcription vectors and translation vectors. Transcription vectors are
utilized when the DNA to be cloned has an ATG start codon and a prokaryotic
ribosome-binding site.
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3.1. Promoters
Proper promoter selection is of the utmost
importance when designing an expression system. In fact, expression vectors
were originally classified by the nature of their promoters because a strong
promoter was considered the most important asset of these vectors (9).
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4.1. Subcellular
Localization
The localization of a protein in the host
cell may affect its production and tertiary structure. Recombinant proteins can
be directed to one of three compartments: cytoplasm, periplasm, or
extracellular medium (secreted).
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Certain vectors and host strains enhance the
likelihood of expressing a soluble protein. One approach to increasing the
soluble yields of aggregated proteins is to improve folding of newly
synthesized proteins through the co-overexpression of cyto-plasmic molecular
chaperones (70).
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The E. coli plasmid vectors available to researchers are
continually fine-tuned, making it easier to express a wide variety of proteins
in any given expression system. A list of commercially available expression
vectors is included in Table 1.
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The Gram-negative bacterium Escherichia coli enjoys widespread use in modern biology as both a model
organism and a microbiological tool. One of the keys to its popularity lies in
the functionality of the lac operon.
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2.1. Expression
of Recombinant Protein
1. Plasmid-bearing
recombinant gene of interest (see Note 1).
2. E. coli host strain (see Subheading
1.3.).
3. LB
growth medium, prepared according to manufacturer's instructions (see Notes 2 and 3).
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3.1. Expression
of Recombinant Protein
1. Grow a
5-mL overnight culture of the expression construct at 37°C with vigorous shaking.
Use the same growth medium as will be used for expression.
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Reporter genes encode easily measurable
traits. Most commonly, they are used to investigate the expression of other
genes for which functional assays are not available or for which measurement of
expressed product is difficult.
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Many of the reporter genes listed in Table 1 are available in different types of vectors that have
been tailored to specific applications. In choosing a vector system, considerations
should be given to several factors.
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2.1. p-Galactosidase Assay
1. PM2
buffer: 36 mM NaH2PO4, 67 mM Na2HPO4,
0.1 mM MgCl2, 2 mM MgSO4
(see Note 1).
2. PM2SH:
Add 135 yL of p-mercaptoethanol to 50 mL of PM2 buffer (see Note 2).
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3.1. p-Galactosidase Assay
1. Grow
cultures of the strains to be assayed. The assay should be performed using
mid-log cultures (see Note 4).
2. Chill
the cultures on ice for at least 10 min, to prevent further growth, and measure
the cell density at OD600.
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1. These
solutions can be autoclaved or filter sterilized. Their performances are not
affected by storage at ambient temperature for up to 6 mo.
2. Prepared
fresh as needed.
3. Keep
cold at approx 4°C.
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Green fluorescent protein (GFP) of the
jellyfish Aqueorea
victoria is a
238-amino-acid, 28-kDa protein that absorbs light with an excitation maximum of
395 nm and fluoresces with an emission maximum of 509 nm (7).
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2.1. Direct
Colony Examination
Short-wavelength UV lamp or appropriate
imaging device.
2.2. Fluorescence
Microscopy
1. Dulbecco's
phosphate-buffered saline (DPBS, pH 7.4).
2.
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3.1. Colony
Examination
This is the quickest and easiest way of visualizing
fluorescent bacterial colonies. In the case of questionable fluorescence, use
the fluorescence microscopy protocol in Subheading 3.2
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1. Grow
cells in the appropriate liquid medium to exponential phase: l x 108
to l x 109 cells/ mL for bacteria, l x 106 to l x 107
for yeast, and l x 104 and l x 105 for mammalian.
2. Mix 5 yL
of liquid culture with 5 yL of molten l% low-melting-point agarose at 37°C.
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