Journal of Bacteriology, February 2000, p. 842-847, Vol. 182, No. 3
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Towards Single-Copy Gene Expression Systems Making Gene
Cloning Physiologically Relevant: Lambda InCh, a Simple
Escherichia coli Plasmid-Chromosome Shuttle System
Dana
Boyd,
David S.
Weiss,
Joseph C.
Chen, and
Jon
Beckwith*
Department of Microbiology and Molecular
Genetics, Harvard Medical School, Boston, Massachusetts 02115
Received 5 August 1999/Accepted 3 November 1999
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ABSTRACT |
We describe a simple system for reversible, stable integration of
plasmid-borne genes into the Escherichia coli chromosome. Most ordinary E. coli strains and a variety of
pBR322-derived ampicillin-resistant plasmids can be used. A single
genetic element, a lambda phage, is the only specialized vector
required. The resultant strains have a single copy of the plasmid
fragment inserted stably at the lambda attachment site on the
chromosome, with nearly the entire lambda genome deleted.
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TEXT |
Because of their high copy number,
pBR322-derived plasmids (3, 19) are widely used in studies
with the bacterium Escherichia coli. The high level of gene
expression from such plasmids is often desirable, for example in the
production of large amounts of a protein for purification purposes.
However, there are also disadvantages to the use of high-copy-number
plasmids. In studies designed to obtain physiologically relevant
measurements or to assess in vivo phenotypes, low-level expression from
a single copy is usually required. Overproduced proteins may generate
phenotypes, including deleterious effects on growth, induction of
stress responses, and altered properties of the protein itself, that
are unrelated to those observed at lower levels of expression. Such
effects can result in misleading quantitative results and incorrect
conclusions about the physiological role and other properties of the
protein under study. In addition, heterogeneity of plasmid copy number within a population can result in a variation in expression level among
single cells. To avoid such complications, a variety of methods for
integrating DNA from plasmids into the chromosome have been employed.
Often these methods involve one or more cloning steps, which can be
time-consuming, particularly when the study involves the construction
of many plasmid derivatives.
We have devised a simple in vivo system for stable chromosomal
integration of the expression systems on many commonly used pBR322-derived plasmids, such as pDHB60 and pDHB5700 (Fig.
1 and Table
1), pUC18 and similar plasmids (18,
23), pTac plasmids such as pKK223-3 (5), pTrc plasmids
(1), pBAD plasmids like pBAD18 (7), and plasmids
expressing green fluorescent protein (GFP) (21). Our system
should be applicable to a wide variety of other pBR322-related plasmid
vectors.
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FIG. 1.
Plasmids on which the lambda InCh vectors are based.
These are suitable cloning vectors for use with lambda InCh1. Both
contain the Tac promoter from pKK223-3 (5).
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General principle.
Our system for generating single-copy
chromosomal versions of plasmid-encoded genes utilizes a bacteriophage
lambda derivative which we have named lambda InCh (for "into the
chromosome"). The transfer requires three successive in vivo steps,
but no in vitro cloning steps. Both homology-dependent recombination
and site-specific recombination are involved. The steps required
for this process are (i) recombination of the desired
plasmid-encoded genes onto lambda InCh; (ii) integration of the
recombinant lambda InCh, carrying the newly incorporated genes, into
the chromosome at the lambda attachment site; and (iii) deletion of
most of the lambda genes, including one attachment site, from the
chromosome of the lysogen (Fig. 2).
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FIG. 2.
The three steps involved in transfer of an expression
system from a pBR322-derived plasmid to the chromosome in stable single
copy. At the upper left, the plasmid and the phage are shown as
concentric circles with the phage outside. In the first step,
recombination at both the 358-bp 'bla region and the 409-bp
near-ori region results in transfer of the promoter gene expression
system and a functional bla (Ampr) gene to the
phage replacing the Kanr gene. In the second step a lysogen
is formed by site-specific recombination at the att site,
conferring ampicillin resistance and temperature sensitivity. The
orientation at the att site is indicated relative to the
flanking markers gal and bio. In the third step,
recombination in the 800-bp direct-repeat region, near-att, removes
most of the phage DNA conferring temperature independence. The
att region in the stabilized strain is shown at the
bottom.
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The first of these recombination events occurs during the growth of the
phage in a cell containing the plasmid of interest. Homologous
recombination at one or the other of two regions shared by the plasmid
and lambda InCh results in cointegrate formation. Resolution of the
cointegrate in a second recombination step at the other region of
homology then results in a transfer of genetic material from the
plasmid to the phage. Cointegrate resolution can occur either during
phage replication in the plasmid-containing cell or at a later stage.
The two homologous regions that allow the double recombination event to
occur are (i) a region near the pBR322 replicative origin, which we
call "near-ori," and (ii) a fragment of the bla
ampicillin resistance gene of pBR322. This fragment, which represents
only half of the gene, does not by itself confer ampicillin resistance,
and the near-ori region, while present in most pBR322-derived vectors,
does not play a role in plasmid replication, nor does it interfere with
subsequent steps. As a result of the double recombination event, the
kanamycin resistance (Kanr) allele of the phage is replaced
both by a complete bla gene, conferring ampicillin
resistance to lysogens, and by the segment between the two regions of
homology. Since the expression systems and the cloned genes on many
pBR322 derivatives are located between near-ori and bla, the
desired region is transferred to the recombinant phage.
Growth of lambda InCh on a strain containing the appropriate plasmid
yields a low-frequency transducing (LFT) lysate. Most of the phages in
this LFT lysate carry the Kanr allele of the parent, but a
minority population, typically 10
3, are recombinant
phages that have the complete bla gene and the desired
expression system replacing the Kanr allele. This LFT
lysate can be used to perform the second step, chromosomal
integration, which involves site-specific recombination between
the lambda attachment sites of the phage (attP) and
E. coli (attB), resulting in a
lysogen. Lambda InCh phages that have picked up the region of interest
will form ampicillin-resistant, kanamycin-sensitive lysogens, in which
the expression system is inserted into the chromosome at the lambda
attachment site, att, as part of the prophage. Induction of
such a lysogen gives, in most cases, a high-frequency transducing (HFT)
lysate, in which all phages carry the bla gene and the
expression system and are clonally derived. Ideally, such a lysate
should be used to select for lysogens in which the expression system is
integrated at the att site.
Although lambda lysogens are stable enough for some purposes, they are
less stable than ordinary chromosomal loci. Such instability can
complicate in vivo studies, such as genetic screens or selections, during which higher-frequency rearrangements involving the prophage may
make it impossible to find desired mutants, which may occur at a lower
frequency. Genetic instability is a consequence of the activity of
prophage genes, which can lead to spontaneous partial induction and the
loss or tandem duplication of the prophage DNA. In addition, multiple
lysogens are often unwittingly obtained when antibiotic resistance is
used to select for lysogens, again posing problems of instability for
the use of such lysogens. For this reason, we designed lambda InCh so
that there is a simple way to select for deletion of most of the lambda
DNA from the lysogens. Lambda InCh vectors were constructed to contain
a fragment of the E. coli chromosome that flanks the
attachment site, which we call "near-att." This property of the
phage results in a situation in which lysogens carry a direct repeat of
DNA, one copy inside and one outside the prophage. At a low frequency,
typically 105, DNA between the direct repeats is deleted
by homologous recombination.
Another property of lambda InCh allows direct selection for derivatives
of lysogenic strains that have deleted all the material between the two
direct repeats. This lambda phage carries the heat-inducible
cI857 repressor, so that induction of the prophage at 42°C
efficiently kills the cell containing the prophage. Since all the
prophage genes responsible for killing the cell lie between the
directly repeated regions, heat induction of lysogens selects for
isolates in which the prophage genes have been lost by a recombination event that occurred in an ancestor of the surviving bacterium. Temperature-independent derivatives are no longer lysogenic and do not
have any of the instability of the lysogens described above. They have
a few bases of the E. coli chromosome near the
att site replaced by a single, stable copy of the inserted
expression system, along with 2.5 kb of DNA from the lambda
b region that has no known phenotype, and a single hybrid
att site.
Lambda InCh-derived chromosomal insertions can be
readily moved from one strain to another by
transduction with phage P1, selecting for ampicillin resistance. The
inserted DNA can also be conveniently recovered from the
chromosome by recombining it onto a high-copy-number plasmid in steps
that are the reverse of those that resulted in its incorporation into
the phage. Simply introducing a suitable recipient plasmid into a donor
strain, preparing plasmid DNA, and transforming, thus selecting for the chromosomal antibiotic resistance, is sufficient (14). This process can be made more efficient by using plasmids containing the M13
intergenic region and by using M13 transduction.
Sample usage of lambda InCh.
We have constructed strains that
carry different copy numbers of alkaline phosphatase to illustrate the
use of lambda InCh. Alkaline phosphatase assay data for lambda InCh
lysogens and stabilized strains are presented in Table
2. The insert from the plasmid in
DHB6046, which contains the malF-phoA fusion J
(4), was picked up on lambda InCh1 and was stabilized on the
chromosome by the methods outlined above. For comparison, data for
strains with known copy numbers of the plasmid insert stabilized on the chromosome by a different method (4) are also presented.
Alkaline phosphatase activity is proportional to copy number in strains with a fixed copy number of the fusion. Plasmid-containing strains have
high activity. A lysogen that was selected in the presence of a high
concentration of ampicillin also has high activity, indicating a
multiple lysogen. After the lysogens were stabilized by selection for
temperature independence, all, including those derived from the
multiple lysogen, had activities consistent with only a single copy of
the insert. Multiple lysogens invariably give rise to single-copy
constructs with this approach because each copy of the prophage carries
the genes which kill the host if they have not been deleted.
Single-copy constructs not only simplify the calculation of units per
copy, as illustrated here, but also minimize toxicity of products
(4).
A second example of the use of lambda InCh is shown in Fig.
3. Here fusions of two cell division
proteins, FtsI and FtsQ, to GFP are shown. The fusions were generated
on a high-copy-number derivative of pTrc99a as described by Weiss et
al. (21). Both strains show extreme heterogeneity in levels
of expression from the plasmid, even without induction. In addition,
both strains have a granular-appearing mislocalization in regions of
the membrane, and the FtsI fusion shows bright polar spots which may be
inclusion bodies. When the genes were stabilized in single copies on
the chromosome, both proteins show septal localization only and much less heterogeneity in expression levels.
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FIG. 3.
Localization of GFP-FtsI (top row) and GFP-FtsQ (bottom
row). The GFP fusion proteins were expressed, without induction, from
the chromosome by using lambda InCh (left column) or from plasmids
(right column). Strains expressing GFP-FtsI and GFP-FtsQ from the
chromosome are EC436 and EC442, respectively (6, 21), while
cells expressing the proteins from plasmids are JOE426 and JOE427,
respectively. Cells were grown at 30°C to early log phase and were
fixed for fluorescence microscopy as described previously
(6).
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Construction of lambda InCh and related plasmids.
Strains,
plasmids, and phages are listed in Table 1. Lambda InCh was constructed
by replacing a 6.5-kb region between the J gene and
attP with a 3.7-kb insert that contains two regions of
pDHB5700 (Fig. 1), the Kanr marker from pUC4K (10,
17), and a chromosomal PCR product. This insert was
constructed in several steps. In step 1, the PstI Kanr fragment of pUC4K was cloned into
PstI-digested pDHB32 (4) to create
pDHB5678, and subsequently the
EcoRI-to-SalI malFG' fragment was
replaced with the EcoRI-to-SalI pUC19
(23) polylinker fragment to make pDHB5700 (Fig. 1). In step
2, the tac promoter of pDHB5700 was deleted by digestion
with BamHI and religation to create pDSW221. In step 3, an 871-bp fragment of E. coli chromosomal DNA derived
from the gal side of attB was cloned into the
DraI site of pDSW221. The fragment was obtained by
using the PCR with JP325 chromosomal DNA as a template and by using two
primers: CCCCTTCAATGTAcaTGTTGGTCACCAGCGTACGCGGCTGACG and
GCAGGCTTCAACatgTTCATTTTTCTATTTCATAGCCC (lowercase bases are mutations that create
AflIII sites). The PCR product was digested with
AflIII, made blunt ended with Klenow fragment in the
presence of all four deoxynucleoside triphosphates, and ligated into
DraI-digested pDSW221. Both orientations of the insert were
recovered. A plasmid with the chromosomal fragment in the desired
orientation (BsiWI site in the fragment proximal to the
AlwNI site in the vector) was designated pDSW222. Finally, in step 4, lambda DNA from New England BioLabs was ligated to join the
cos sites, digested with SacI, treated with T4
DNA polymerase to make the SacI ends blunt, and digested
with BsiWI. This deleted 6.5 kb. This lambda DNA was then
ligated with a 3.5-kbp AflIII-BsiWI fragment from
pDSW222 (which includes the 1.3-kb Kanr fragment). (The
AflIII ends were made blunt by T4 DNA polymerase in
the presence of four deoxynucleoside triphosphates.) The ligation mixture was packaged with Gigpack Gold packaging extract (Stratagene). A stationary-phase culture of SM551 was infected, incubated for 45 min
at 30°C, and plated onto NZ, selecting for resistance to 40 mg of
kanamycin per ml. The resulting phage, which is shown in Fig. 2 as the
outer circle at the upper left, is 3 kb smaller than lambda, including
a 1.3-kb Kanr fragment that is replaced by a plasmid insert
during use. Since there is room for an additional 5% in excess of the
wild-type genome size (2.4 kb) in lambda, the theoretical maximum
insert size is 6.7 kb.
Lambda InCh2 was constructed in two steps, starting with pUC4K
(10, 17). First a deletion from the StuI site at
bp 3405 (across 0/3914) to the ScaI site at 477 was made.
The kanamycin resistance allele of the resulting plasmid was then
recombined onto an ampicillin-resistant derivative of lambda InCh1 (a
typical derivative in which a complete bla gene and
expression system had been recombined onto the phage). This in vivo
step, which relied on 117 bp of homology between pUC plasmids and
pBR322 in the near-ori region and a 600-bp 'bla fragment,
was much less efficient than pBR322-lambda InCh1 recombination. Lambda
InCh2 has about 400 bp of homology to pUC18 in the near-ori region and is much more efficient than lambda InCh1 in incorporation of expression cassettes from pUC-type plasmids. In addition, it is missing an inappropriate segment of homology which would lead to undesired recombination if lambda InCh1 were used with a pUC plasmid.
pDHB60 (Fig. 1) was constructed by cloning the pUC12 (11,
18) polylinker into pDHB32 (4), using the unique
EcoRI and HindIII sites.
Technical details for working with lambda InCh.
The lambda
InCh vectors described here carry the cI857 repressor
mutation and an amber mutation in gene S. Lysogens are
therefore temperature sensitive and can be safely grown at temperatures below about 35°C. S amber mutants of lambda cannot form
plaques on nonsuppressing hosts but, when induced in nonsuppressing
hosts, produce phage which can be released by treatment with
chloroform. Such phages are viable on suppressing hosts and can be used
to select lysogens on both suppressing and nonsuppressing hosts. Induction of lambda InCh lysogens is accomplished by standard methods
(2). Briefly, a culture in broth with 1 mM MgSO4
is grown to log phase at 30°C and is incubated at 42°C for 15 min and then at 37°C until lysis (about 1 h later in a suppressing host), or until the addition of 0.01 volume of chloroform (about 3 to
5 h later in a nonsuppressing host).
The double-recombination event required for transferring the plasmid
genes onto the phage can occur during growth of the phage (recombination frequency is high during growth of lambda [12, 15]), but all that is required for transduction is
cointegration by a single recombination event. Since LFT lysates
contain predominantly parental phage, recombinant lysogens obtained at
a high multiplicity of infection are often double lysogens which also
contain the parental prophage. When stabilized in single copies in the
subsequent steps, such lysogens often yield only the parental type. To
avoid such double lysogens, it is important to employ serial dilutions of the lysate and to pick from the highest-dilution plate that has
colonies. Besides containing double-recombinant phage in which the
plasmid insert has replaced that of the parental phage, LFT lysates
contain packaged cointegrate molecules. Most of these are resistant to
both antibiotics and can immediately be eliminated by screening, but a
few of them are homogenotized cointegrates, resistant to ampicillin
only. After lysogenization, resolution of cointegrates is favored
because insertion of a plasmid origin into the chromosome is
deleterious (22). HFT lysates made from such lysogens are
likely to have a small fraction of reformed cointegrate phage. These
can present a problem if there is some selection favoring
plasmid-containing strains in subsequent steps. (For example, the
bla gene of pDHB60 and pBAD plasmids in single copies gives
resistance to only 0.025 mg of ampicillin per ml. Selection at higher
concentrations results in either multiple lysogens or transduction of
plasmid.) This can be avoided by making a secondary lysogen, using the
primary HFT lysate and preparing a secondary HFT lysate from that. This
should reduce the likelihood that the lysate contains cointegrate
phage, unless there is some systematic problem. (For example, if there
is only one region of homology, only cointegrates will transduce
ampicillin resistance.)
The nominal size limit for DNA packaged in lambda is 105% of the
genome size (9). This would allow for a total of about 7 kb
of insert in lambda InCh vectors. Of this, about 1 kb comprises the
homology regions used for recombination, so about 6 kb is the nominal
limit for insert size. The nominal limit is not an absolute limit,
however (16). We have successfully used plasmids with up to
7.8 kb total inserted in lambda vectors similar to those described here
(H. Tian, D. Boyd, and J. Beckwith, unpublished data). Phage particles
with more than the nominal limit are less stable. By using lysates
immediately, or even by mixing the recipient with the donor culture
during lysis, it is possible to work with larger inserts.
A manual containing detailed protocols for working with lambda InCh and
software to aid in experimental design are available at
http://rcc.med.harvard.edu/~dboyd.html and
http://beck2/resources/InCh/Lambda_InCh.html.
Ease of use and efficiency.
Other methods for inserting DNA
into the chromosome exist (6, 13). Some of these are for use
with fusion proteins and involve plasmid recombination with
bacteriophage lambda (14, 20). Some of these methods produce
a final result that is similar to that achieved here, a stable
nonlysogen with a single copy of the expression system on the
chromosome (24). The advantages of our system lie in its
simplicity. Many genes have already been cloned in expressions systems
compatible with lambda InCh vectors and can therefore be used
immediately. Insertion onto the chromosome is usually not the primary
concern of someone carrying out gene expression. The primary goals of
obtaining the correct construct and ensuring expression of the correct
product are best achieved using a well-characterized, high-copy-number
expression system like those compatible with lambda InCh vectors.
However, for physiological experiments, expression from
high-copy-number systems can be deleterious. The lambda InCh approach
simplifies the subsequent process of obtaining a stable, single-copy
chromosomal insertion so that physiologically relevant experiments can
much more easily be done.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-1920. Fax: (617)
432-7664. E-mail: jbeckwith{at}hms.harvard.edu.
Present address: Department of Microbiology, University of Iowa,
3403 Bowen Science Building, Iowa City, IA 52242.
| |
REFERENCES |
| 1.
|
Amann, E.,
B. Ochs, and K. J. Abel.
1988.
Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli.
Gene
69:301-315[CrossRef][Medline].
|
| 2.
|
Arber, W.,
L. Enquist,
B. Hohn,
N. Murray, and K. Murray.
1983.
Experimental methods for use with lambda, p. 433-466.
In
R. W. Hendrix, J. W. Roberts, F. Stahl, and R. A. Weisberg (ed.), Lambda II. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 3.
|
Bolivar, F.,
R. L. Rodriguez,
P. J. Greene,
M. C. Betlach,
H. L. Heyneker, and H. W. Boyer.
1977.
Construction and characterization of new cloning vehicles. II. A multipurpose cloning system.
Gene
2:95-113[Medline].
|
| 4.
|
Boyd, D.,
C. Manoil, and J. Beckwith.
1987.
Determinants of membrane protein topology.
Proc. Natl. Acad. Sci. USA
84:8525-8529[Abstract/Free Full Text].
|
| 5.
|
Brosius, J., and A. Holy.
1984.
Regulation of ribosomal RNA promoters with a synthetic lac operator.
Proc. Natl. Acad. Sci. USA
81:6929-6933[Abstract/Free Full Text].
|
| 6.
|
Chen, J. C.,
D. S. Weiss,
J.-M. Ghigo, and J. Beckwith.
1999.
Septal localization of FtsQ, an essential cell division protein in Escherichia coli.
J. Bacteriol.
181:521-530[Abstract/Free Full Text].
|
| 7.
|
Gutterson, N. I., and D. E. Koshland, Jr.
1983.
Replacement and amplification of bacterial genes with sequences altered in vitro.
Proc. Natl. Acad. Sci. USA
80:4894-4898[Abstract/Free Full Text].
|
| 8.
|
Guzman, L.-M.,
D. Belin,
M. J. Carson, and J. Beckwith.
1995.
Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.
J. Bacteriol.
177:4121-4130[Abstract/Free Full Text].
|
| 9.
|
Murray, N.
1983.
Phage lambda and molecular cloning, p. 395-431.
In
R. W. Hendrix, J. W. Roberts, F. Stahl, and R. A. Weisberg (ed.), Lambda II. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 10.
|
Oka, A.,
H. Sugisaki, and M. Takanami.
1981.
Nucleotide sequence of the kanamycin resistance transposon Tn903.
J. Mol. Biol.
147:217-236[CrossRef][Medline].
|
| 11.
|
Pouwels, P. H.,
B. E. Enger-Valk, and W. J. Brammar.
1985.
Cloning vectors.
Elsevier, New York, N.Y.
|
| 12.
|
Signer, E.
1971.
General recombination, p. 139-174.
In
A. D. Hershey (ed.), The bacteriophage lambda. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 13.
|
Silhavy, T. J., and J. R. Beckwith.
1985.
Uses of lac fusions for the study of biological problems.
Microbiol. Rev.
49:398-418[Free Full Text].
|
| 14.
|
Simons, R. W.,
F. Houman, and N. Kleckner.
1987.
Improved single and multicopy lac-based cloning vectors for protein and operon fusions.
Gene
53:85-96[CrossRef][Medline].
|
| 15.
|
Smith, G.
1983.
General recombination, p. 175-210.
In
R. W. Hendrix, J. W. Roberts, F. Stahl, and R. A. Weisberg (ed.), Lambda II. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 16.
|
Struck, D. K.,
D. S. Durica, and R. Young.
1986.
Gene fusion in vivo using transductional cointegrates.
Gene
47:221-230[CrossRef][Medline].
|
| 17.
|
Taylor, L. A., and R. E. Rose.
1988.
A correction in the nucleotide sequence of the Tn903 kanamycin resistance determinant in pUC4K.
Nucleic Acids Res.
16:358[Free Full Text]. (Erratum, 16:7762.)
|
| 18.
|
Vieira, J., and J. Messing.
1982.
The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers.
Gene
19:259-268[CrossRef][Medline].
|
| 19.
|
Watson, N.
1988.
A new revision of the sequence of plasmid pBR322.
Gene
70:399-403[CrossRef][Medline].
|
| 20.
|
Weisemann, J. M.,
C. Funk, and G. M. Weinstock.
1984.
Measurement of in vivo expression of the recA gene of Escherichia coli by using lacZ gene fusions.
J. Bacteriol.
160:112-121[Abstract/Free Full Text].
|
| 21.
|
Weiss, D. S.,
J. C. Chen,
J.-M. Ghigo,
D. Boyd, and J. Beckwith.
1999.
Localization of FtsI (PBP3) to the septal ring requires its membrane anchor, the Z ring, FtsA, FtsQ, and FtsL.
J. Bacteriol.
181:508-520[Abstract/Free Full Text].
|
| 22.
|
Yamaguchi, K., and T. Tomizawa.
1980.
Establishment of Escherichia coli cells with an integrated high copy number plasmid.
Mol. Gen. Genet.
178:525-533[CrossRef][Medline].
|
| 23.
|
Yanisch-Perron, C.,
J. Vieira, and J. Messing.
1985.
Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors.
Gene
33:103-119[CrossRef][Medline].
|
| 24.
|
Yu, D., and D. Court.
1998.
A new system to place single copies of genes, sites and lacZ fusions on the Escherichia coli chromosome.
Gene
223:77-81[CrossRef][Medline].
|
Journal of Bacteriology, February 2000, p. 842-847, Vol. 182, No. 3
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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