Gene 187 (1997) 231–238
A method for construction of E. coli strains with multiple DNA
insertions in the chromosome
Michael Y. Peredelchuk, George N. Bennett *
Department of Biochemistry and Cell Biology, Rice University, 6100 S. Main Street, Houston, TX 77005-1892, USA
Received 20 May 1996; revised 16 September 1996; accepted 3 October 1996; Received by A.M. Campbell
Abstract
A system for construction of E. coli strains with multiple DNA insertions in the chromosome, based on elements of modules
for site specific recombination of Tn1545 and phage l, has been developed. Circular non-replicating DNA fragments containing
the transposon attachment site (attTn), an excisable cassette with a selectable marker, and a gene of interest integrate randomly
into the chromosome of a host E. coli strain when provided with transposon integrase, Int-Tn (the host strain was obtained by
insertion of the fragment containing transposon int-Tn gene coding for Int-Tn into the chromosome). Integration of these
fragments into the chromosome of int-Tn+ cells gives rise to a collection of antibiotic-resistant clones with single insertions at
different locations in the chromosome. These insertions are transferred subsequently by P1 transduction into one strain and
selected for antibiotic resistance provided by the cassette with the selectable marker. After transduction of each copy, a helper
plasmid bearing phage l xis and int genes is introduced into the cells to excise the drug resistance gene flanked with the lattL
and lattR sites from the chromosome. Cells cured of the helper plasmid can undergo the next cycle of P1 transduction/drug
resistance gene excision. Each cycle adds another chromosomal copy of the foreign gene. To show the utility of the system, we
constructed an E. coli strain bearing several chromosomal copies of lacZ at different locations.
Keywords: Amplification; Site-specific recombination; Tn1545; Phage l; Integration; Excision; P1 transduction
1. Introduction
The industrial use of genetically engineered bacteria
depending on the presence of autonomously replicating
* Corresponding author. Tel. +1 713 5274920; Fax +1 713 2855154;
e-mail: [email protected]
Abbreviations: aph, KmR-encoding gene; lattB and lattP, bacterial
and phage l attachment sites, respectively; lattL and lattR, left and
right boundaries of prophage l, respectively; attTn, Tn1545 attachment site; bGal, b-galactosidase; bla, ApR-encoding gene; bp, base
pair(s); cat, CmR-encoding gene; cIts857, gene encoding thermosensitive repressor of phage l; Cm, chloramphenicol; E., Escherichia;
Int-Tn, Tn1545 integrase; int-Tn, Int-Tn-encoding gene; IPTG, isopropyl-b--thiogalactopyranoside; kb, kilobase pair(s) or 1000 bp; Km,
kanamycin; lacI, lac repressor-encoding gene; lacZ, bGal-encoding
gene; lacZpo, promoter-operator of E. coli lactose operon; LB, LuriaBertani medium; lInt, phage l integrase; lint, lInt-encoding gene;
lXis, phage l excisionase; lxis, lXis-encoding gene; MCS, multiple
cloning site; mrs, multimer resolution system; OD, optical density; ori,
origin of replication; PCR, polymerase chain reaction; p , major early
R
rightward phage l promoter; R, resistant/resistance; S,
sensitive/sensitivity; Tc, tetracycline; tet, TcR-encoding gene; Tn,
transposon; Xis-Tn, Tn1545 excisionase; xis-Tn, Xis-Tn-encoding
gene; ∞, designates gene truncation.
plasmids has some drawbacks. A major one is the
difficulty in maintaining these plasmids without loss due
to their segregational instability. The problem is mostly
solved by introducing antibiotic resistance genes into
plasmids. Another problem is rearrangements and deletions within expression plasmids. Loss of the genes to
be expressed often imparts a cell growth advantage, and
these cells will rapidly overgrow their parents. The
pressures exerted on autonomous replicons appear to
be absent or reduced when genes are carried as fragments
inserted into the bacterial chromosome. Therefore, integration of DNA into bacterial genomes would be a
feasible approach for the stabilization of heterologous
genes in industrially important bacteria.
In circumstances related to metabolic engineering,
considerations of cost, stability and optimal relative
activity of enzyme of particular pathway are important
concerns, rather than just high production of a specific
single protein. In these applications a technique allowing
one to obtain multiple chromosomal insertions may
provide an alternative route to the construction of
useful strains.
0378-1119/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved
PII S 03 7 8 -1 1 1 9 ( 9 6 ) 0 0 7 60 - 3
232
M.Y. Peredelchuk, G.N. Bennett / Gene 187 (1997) 231–238
Fig. 1. The principle of the method. (1) Circular non-replicating DNA fragments containing attTn, an excisable cassette with a selectable marker,
and a gene of interest integrate randomly into the chromosome of a host E. coli int-Tn+ strain giving rise to a collection of antibiotic-resistant
clones with single insertions at different locations in the chromosome. (2) These insertions are transferred subsequently by P1 transduction into
an int-Tn− strain with selection for antibiotic resistance provided by the cassette. (3) After transduction of each copy, a helper plasmid bearing
phage l xis and int genes is introduced into the cells to excise the drug resistance gene flanked with lattL and lattR from the chromosome. (4)
Cells are cured of the helper plasmid and can undergo the next cycle of P1 transduction/drug resistance gene excision. XYZ, foreign DNA.
Integration of multiple copies of heterologous genes
into bacterial genomes, required for high-level expression, can be performed in several ways. One of them is
a single insertion into the chromosome of a DNA
fragment containing a drug resistance marker, flanked
by short direct repeats, with further amplification of the
entire fragment in response to increasing antibiotic
concentration. However, sequences amplified in such a
way are often unstable, and antibiotics are still needed
to maintain the integrity of the amplified structures in
the resulting strains (Mori et al., 1988; Chopin et al.,
1989).
Another approach is integration of DNA fragments
at multiple locations in the chromosome using homologous recombination (Pósfai et al., 1994; Kiel et al.,
1995). This method requires construction of a number
of individual integrative plasmids to perform the insertion of each copy. Another drawback of this approach
might be the comparatively low efficiency of homologous
recombination in some bacteria, which makes it difficult
to perform allelic exchange.
Here we describe a system for construction of E. coli
strains with multiple insertions of DNA in the chromosome based on elements of modules for site specific
recombination of Tn1545 and phage l.
2. Materials and methods
2.1. Strains, plasmids and media
The following E. coli strains, plasmids, phages and
media were used in this work: E. coli strains – XL1-blue
MRF ∞ (Stratagene, La Jolla, CA, USA) and LE392
(Murray et al., 1977); plasmids – pACT7 (Maksimova
et al., 1991), pACYC184 (Chang and Cohen, 1978),
M.Y. Peredelchuk, G.N. Bennett / Gene 187 (1997) 231–238
233
2.3. Integration of int-Tn into the E. coli chromosome
Fig. 2. Integrative plasmid pMP951 consists of: the SphI-ScaI fragment
(567–3848 bp) of pBR322; the part of MCS of pTZ19RJL1 from
HincII to EcoRI (282–335 bp) derived from the insertion, into the
BamHI site, of the fragment bearing cat and obtained by cleavage with
BamHI of the PCR fragment amplified using pSG335 as a template
and oligonucleotides 5∞-CGTAAGAGGATCCAACTTTCACC-3∞ and
5∞-CCGGATCCAAAAACTTCAAGTATATGAAAGATCTCTTATCCTTGAAGAAAAAGGCCTAGCACCAGGCGTTTAAGG-3∞ as
primers and including the insertion, into the MluI site, of the attTncontaining fragment obtained by cleavage with MluI of the PCR fragment amplified using pAT113 as a template and oligonucleotides
5∞-TTTGGAAAGTTACGCGTTACTAAAGGGAATGG-3∞ and 5∞CGGCCTTCTTAAAGACGCGTAAAGAAAAATAAGCAC-3∞ as
primers; the EcoRI-BamHI fragment of plasmid p1002 bearing cIts857
and p ; the fragment containing the sequence of int-Tn obtained by
R
cleavage with BamHI and HindIII of the fragment amplified by PCR
using pAT295 as a template and oligonucleotides 5∞-TGGATCCATAAAGGAAAGGAGCAATTGCCATGTCAG-3∞ and 5∞AATTCATTTGTAAGCTTAAGCAACAAGACGCTCCTG-3∞
as
primers; the HindIII-PvuII fragment (266–56 bp) of pTZ19RJL1; the
fragment of MCS of pUC18 from SmaI to SphI (433–402 bp). Af,
AflII; Ap, ApaI; B, BamHI; E, EcoRI; H, HindIII; Hc, HincII (shown
only when used for cloning); K, KpnI; M, MluI; Nc, NcoI; P, PstI;
Pv, PvuII; S, SalI; Sc, ScaI; Sm, SmaI; Sn, SnaI; Sp, SphI; X, XbaI.
Asterisk shows sites in the MCS that are not unique.
pAT113 ( Trieu-Cuot et al., 1991), pAT295 (PoyartSalmeron et al., 1989), pBluescriptSK+ (Stratagene),
pBR322 (Bolivar et al., 1977), pINTK (Peredelchuk
et al., 1994), pRS552 (Simons et al., 1987), pSG335
(Geissler and Drummond, 1993), pTZ19RJL1
(Fermentas MBI, Vilnius, Lithuania), pUC4K ( Vieira
and Messing, 1982), pUC18 ( Yanisch-Perron et al.,
1985), p1002 (Hartley, R.W., personal communication,
1993); media – LB broth and agar (Miller, 1972).
2.2. Recombinant DNA techniques, P1 transduction and
bGal activity assay
Standard DNA procedures were carried out according
to published protocols (Sambrook et al., 1989). P1
transduction and bGal activity assay were performed as
described (Miller, 1972).
To integrate int-Tn into the chromosome of E.
coli, pMP951 (Fig. 2) was cleaved with PstI and the
fragments were separated by agarose gel electrophoresis
to remove ori. The fragment containing cat, attTn, and
the cIts857-p -int-Tn cassette was eluted from the gel
R
and ligated. Non-replicating circular DNA molecules
were used to transform XL1-blue MRF ∞ cells. After
heat shock the cells were incubated for 1 h at 37°C in
LB broth and plated on LB agar containing Cm.
Integrants were tested for the presence of int-Tn in the
chromosome by Southern blot hybridization and for
their ability to provide integration into the chromosome
of non-replicating circular DNA molecules bearing
attTn.
2.4. Integration of single copies of lacZ at different sites
into the E. coli chromosome
To integrate the cIts857-p -lacZ cassette into the E.
R
coli chromosome at different locations, pMP957
( Fig. 5B) was cleaved with XbaI to remove ori, the
originless fragment was separated by agarose gel electrophoresis, eluted from the gel, and self-ligated to give
non-replicating circular DNA molecules. The circular
DNA obtained was used to transform int-Tn+ host cells.
Transformed cells were incubated at 37°C. Integrants
were selected for KmR. The presence of single insertions
at different locations in the chromosome was confirmed
by Southern blot analysis.
3. Results and discussion
3.1. Principle of the method
The general idea behind the scheme is to use a pair
of systems for site specific recombination: one of them
with low site specificity to achieve random integration
of the gene of interest and a drug-resistant marker into
the chromosome and the second one with stringent
specificity for recombination sites to carry out excision
of the drug marker from the chromosome.
Our system makes use of modules for site-specific
recombination of phage l and Tn1545. Integration of
phage l into the chromosome, which is a result of
recombination between the lattP and lattB sites, is
provided by lInt (Nash et al., 1977). Effective excision
of the prophage from the chromosome, flanked there
by lattL and lattR, is dependent upon action of lXis
and lInt proteins ( Echols and Guarneros, 1983). We
used lattL and lattR for construction of a cassette
consisting of a drug resistance gene flanked with these
sites. The cassette allows selection of clones with inser-
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M.Y. Peredelchuk, G.N. Bennett / Gene 187 (1997) 231–238
Fig. 3. (A) lattB-bearing plasmid pMP952A was constructed by cloning the DNA fragment obtained by cleavage with EcoRI and BamHI of a
PCR fragment amplified using pSG335 as a template and oligonucleotides 5∞-AAATGAATTCGTTGAAGCCTGCTTTTTTATACTAACTTGAGCGAAAGG-3∞ and 5∞-CCGGATCCAAAAACTTCAAGTATATGAAAGATCTTTATCCTTGAAGAAAAAGGCCTAGCACCAGGCGTTTAAGG-3∞ as primers, into EcoRI and BamHI cleaved (2–1873 bp) pACYC184 giving pMP952. During the cloning deletion of the EcoRI
site occurred and resulted in the following sequence at the junction between pACYC184 (2 bp) and the PCR fragment (23 bp): (pACYC184) 5∞…
TCATCCGG/TTTTTTATA…3∞ (lattB). However, the modified lattB proved functional. (B) pMP952B consists of the following fragments: the
HincII-Ecl136II fragment of pUC18 (263–238 bp), the DraI-StuI fragment of pMP952A containing lattB, the HincII fragment of pUC4K containing
aph, and the NheI-StuI fragment of pMP952A containing lattB. (C ) lattL and lattR were generated by site-specific recombination between lattB
and lattP of pMP952 and pINTK, respectively, to give pMP953. D. pMP954 bears the excisable drug-resistant cassette as an insert into SnaI and
HindIII sites of the MCS of pTZ19RJL1. The cassette consists of the following fragments of pMP953: the SnaI-NcoI fragment bearing lattL, the
SalI-StuI fragment bearing aph and lattR, and the StuI-HindIII fragment. B, BamHI; D, DraI; E, EcoRI; H, HindIII; Nc, NcoI; Nh, NheI; S, SalI;
Sn, SnaI; St, StuI; V, EcoRV; X, XbaI.
tions of DNA fragments into the chromosome and can
be excised upon integration when provided with lXis
and lInt proteins.
For integration of DNA at multiple locations into the
chromosome, we chose to exploit elements of the conjugative transposon Tn1545 which has an enormously
wide host range (Clewell et al., 1995). Transposition of
Tn1545 proceeds by excision of a free non-replicating
covalently closed intermediate which is a substrate for
integration. The integration-excision system of the
transposon is structurally and functionally related to
that of lambdoid phages (Poyart-Salmeron et al., 1989,
1990). The products of xis-Tn and int-Tn function
similarly to those of phages, with Xis-Tn and Int-Tn
acting at the coupling sequences to facilitate effective
excision, while only Int-Tn is needed for integration
(Poyart-Salmeron et al., 1989). Int-Tn was shown able
to act in trans. Tn1545 and its derivatives do not exhibit
apparent preference for a particular target sequence and
integrate into AT-rich regions bearing short segments
of partial homology with the ends of the transposon
(Poyart-Salmeron et al., 1990; Trieu-Cuot et al., 1991).
By analogy with the recombination system of bacteriophage l, the att sites of Tn1545 and of the targets were
designated attTn and attB, respectively ( Trieu-Cuot
et al., 1993). These properties of the transposon allow
use of its elements for integration at multiple locations
in the bacterial chromosome: non-replicating circular
M.Y. Peredelchuk, G.N. Bennett / Gene 187 (1997) 231–238
235
Fig. 4. (A) The helper plasmid pMP955A was constructed by insertion of the NdeI-SspI fragment of lDNA (27631–29156 bp) (Daniels et al.,
1983) containing lxis and lint into pUC18 cleaved with NdeI and Ecl136 II. (B) pMP955B was constructed by insertion of the PvuII-NdeI fragment
of pMP565A into pACYC184 cleaved with HincII and VspI. Ec, Ecl136 II; Nd, NdeI; Ss, SspI; Vs, VspI.
DNA molecules containing attTn will integrate into the
chromosome when provided with Int-Tn.
The integrative plasmid designed for this system contains the gene of interest, attTn providing for integration
into the chromosome, and the excisable drug marker
cassette for selection of clones in which integration into
the chromosome has occurred ( Fig. 1). The originless
circularized fragment of the integrative plasmid is used
for transformation of the cells of the host E. coli strain
which contains a chromosomal copy of int-Tn under the
control of an inducible promoter. Integrants resulting
from insertion of DNA circles into the chromosome are
selected for drug resistance conferred by the selectable
marker. Thus, a collection of antibiotic-resistant clones
with single insertions at multiple locations in the chro-
mosome can be obtained. Inserts are transferred subsequently by P1 transduction into one strain and selected
for antibiotic resistance provided by the cassette with
the selectable marker. Upon transduction of each copy,
a helper plasmid bearing lxis and lint is introduced into
the cells. When expressed, these genes provide production of lXis and lInt which excise the drug resistance
gene flanked with lattL and lattR from the chromosome. Cells cured of the helper plasmid can undergo the
next cycle of P1 transduction/drug resistance gene excision, which would add another chromosomal copy of
the foreign gene. Thus, the system allows construction
of a strain with multiple insertions of foreign genes into
the chromosome.
Fig. 5. (A) The integrative vector pMP956 was constructed by insertion of the EcoRV-DraI fragment of pMP954 into the EcoRV site of the MCS
of pBluescriptSK+, and of the fragment containing the sequence of attTn obtained by cleavage with EcoRI and BamHI of the PCR fragment
amplified using pAT113 as a template and oligonucleotides 5∞-CCCGGGAATTCGGATAAATCGTCGTATCAAAGCTCT-3∞ and 5∞-CCCGGGATCCAGGAGCGTCTTGTTGCTTAGTAGTTAG-3∞ as primers into the EcoRI and BamHI sites of the MCS of pBluescriptSK+. (B) The integrative
plasmid pMP957 for obtaining multiple insertions of lacZ in the chromosome consists of the following parts: the BsaAI-BamHI fragment of
pACT7, the BamHI-DraI fragment of pRS552, the part of synthetic polylinker, the SalI-BsaAI fragment of pACYC184 (2146–3143 bp), and the
StuI-Ecl136 II fragment of pMP956. Ap, ApaI; B, BamHI; Bs, BsaAI; Bt, BstXI; C, ClaI; D, DraI; E, EcoRI; H, HindIII; M, MluI; N, NotI;
S, SalI; Sa, SacII; Se, SpeI; Sl, SplI; St, StuI; V, EcoRV; X, XbaI; Xh, XhoI. Asterisk shows sites in the MCS that are not unique.
236
M.Y. Peredelchuk, G.N. Bennett / Gene 187 (1997) 231–238
replicating circularized fragment into the chromosome
of E. coli. The resulting int-Tn+ strain served as a host
that could provide integration of any non-replicating
circular DNA bearing attTn into different loci on the
chromosome upon thermoinduction of the chromosomal
copy of int-Tn.
3.3. Construction of an excisable cassette with a
selectable marker
Fig. 6. Southern blot analysis of LE392 derivatives carrying single
and multiple copies of lacZ. Genomic DNA of LE392 ( lane 1) and
its derivatives with 1, 2, and 3 chromosomal insertions of the
cIts857-p -lacZ cassette ( lanes 2, 3, and 4, respectively) corresponding
R
to those in Table 1 was digested with EcoRV and hybridized with the
internal EcoRV-DraI fragment of lacZ (2413–4421 bp) ( Kalnins et al.,
1983) as a probe.
3.2. Construction of an E. coli strain providing
integration of circular DNA bearing attTn into different
loci on the chromosome
Previously a system for integration of single copies of
foreign genes into the chromosome of Gram-positive
bacteria, based on elements of Tn1545, was reported
( Trieu-Cuot et al., 1991). It contained an integrative
plasmid bearing attTn and a helper plasmid bearing intTn. As Int-Tn was shown to catalyze excision of Tn1545
derivatives at low rates even in the absence of Xis-Tn
(Poyart-Salmeron et al., 1989), the system containing
int-Tn and attTn in trans allows one to obtain more
stable insertions in the chromosome because the insert
can be transferred upon integration into an int-Tn−
strain. In the system described in that report int-Tn was
placed under the control of an inducible promoter on
the helper plasmid. Taking into consideration that
Tn1545 and its derivatives integrate more or less randomly, we were alerted that the presence of a helper
plasmid might lead to recombination between the plasmid and attTn-bearing molecules and additional efforts
would be necessary to distinguish between clones bearing
inserts in the chromosome and clones with inserts in the
plasmid. To avoid recombination with the helper plasmid, we inserted int-Tn into the chromosome of a strain
to be used as a host for integration. To reach this goal,
we constructed an integrative plasmid pMP951 bearing
int-Tn and attTn in cis (Fig. 2) and inserted its non-
Systems for integration of single copies of foreign
genes into the chromosome usually include drug-resistance markers which allow selection of the integrants.
However, multiple insertions in the chromosome of the
same strain can exhaust availability of drug markers,
especially for species with only few antibiotic resistance
genes available. Introduction of multiple insertions into
the chromosome would also require construction of a
new integrative plasmid for integration of each DNA
fragment and would lead to construction of strains with
multiple antibiotic resistance. Such strains may be unacceptable for use in environmental applications or as live
vaccines (De Lorenzo and Timmis, 1994; Cirillo et al.,
1995).
The use of cassettes bearing a drug selectable marker
which could be excised from the chromosome upon
integration would make it possible to overcome these
limitations. Recently, a number of excisable cassettes
with antibiotic resistance genes have been described,
based on elements of different systems for site-specific
recombination: Cre/lox of phage P1, Flp/FRT of 2 mm
yeast plasmid, and mrs system of plasmid RP4 (Sauer,
1994; Pósfai et al., 1994; Cherepanov and Wackernagel,
1995; Kristensen et al., 1995). All these systems do not
require any host factors for recombination and, therefore, can be used in a variety of prokaryotic and
eukaryotic hosts. Both integration and excision are
mediated by enzymes belonging to the integrase family
and do not require Xis-like proteins. These systems,
however, would create inconvenience were they to be
used for construction of strains with multiple insertions
in the chromosome. As a resistance determinant
removed from the chromosome leaves behind a short
sequence with an active site for recombination, these
sites would be amplified in the chromosome as a result
of multiple insertions and could subsequently interact
with each other causing chromosomal rearrangements
and deletions.
Due to these concerns, we decided to use elements of
the module for site specific recombination of phage l
for construction of the excisable selectable cassette. The
construction of the cassette involved the following steps:
construction of the plasmid pMP952A containing lattB
( Fig. 3A), formation of the plasmid pMP953 bearing
lattL and lattR by site-specific recombination between
lattB and lattP ( Fig. 3C ), and construction of pMP954
237
M.Y. Peredelchuk, G.N. Bennett / Gene 187 (1997) 231–238
harbouring the cassette consisting of aph flanked with
lattL and lattR ( Fig. 3D). Excision of the drug marker
is provided by the helper plasmid pMP955A ( Fig. 4A)
bearing lxis and lint under the control of an inducible
promoter. Thus, the selectable drug resistance marker
can be excised from the chromosome leaving behind a
short sequence with lattB. As the presence of lattP is
required for recombination with lattB, multiple insertions of lattB sites should be unable to interact with
each other in the chromosome.
To test the stability of DNA bearing multiple lattB
we constructed the plasmid pMP952B where aph is
flanked with similarly oriented lattB sites ( Fig. 3B). In
the presence of the helper plasmid pMP955B ( Fig. 4B),
after 3 h of incubation upon induction of lxis and lint,
we could not detect loss of the aph gene flanked by the
lattB. Under similar conditions, upon induction of flp,
90% of the plasmids containing a fragment flanked by
a pair of directly oriented FRT sites were reported to
lose the fragment (Pósfai et al., 1994).
3.4. Integration of multiple copies of lacZ into the
E. coli chromosome
To construct integrative vector pMP956, we inserted
attTn and the cassette with an excisable drug marker
into pBluescriptSK+ ( Fig. 5). The resulting vector contains a module consisting of attTn and an excisable drug
marker and can be used for integration of subcloned
foreign DNA fragments into the chromosome. Another
approach to obtain an integrative plasmid is to subclone
the module in the plasmid bearing the gene of interest.
To show the utility of the system, we used lacZ under
the control of cIts857 and p for insertion into the
R
chromosome at different locations. To construct an
integrative plasmid pMP957, we combined together the
cIts857-p -lacZ module, the excisable cassette with aph,
R
and attTn ( Fig. 5). The originless circularized fragment
of the plasmid was used to transform int-Tn+ E. coli
cells. Thus, a collection of KmR clones with an insertion
of the fragment at multiple locations in the chromosome
was obtained.
Then, P1 transduction was used to transfer chromosomal inserts into one strain. It was shown that Int-Tn
can catalyze excision of Tn1545 even in the absence of
Xis-Tn (Poyart-Salmeron et al., 1989). To avoid IntTn-mediated disintegration of the obtained inserts, we
transferred them into int-Tn− LE392. After transfer of
each copy and selection of the transductants for KmR,
cells were transformed with pMP955 bearing lint and
lxis. Provided with lXis and lInt, aph flanked with
lattL and lattR was excised from the chromosome.
Cells were cured from the helper plasmid and transduction of the next copy was performed. Repeating these
cycles, we transferred three copies of lacZ into the
Table 1
bGal activity of E. coli LE392 derivatives with chromosomal insertions
of lacZ (see Fig. 6)
Number of integrated lacZ copies
bGal activity (Miller units)
0
1
2
3
~0
270
390
560
Cells were grown in LB broth at 30°C to OD
0.9–1.0. To express
600
the integrated copies of lacZ under the control of thermoinducible o
, cells were incubated at 37°C for 2 h. Since the medium did not
R
contain IPTG, the contribution of the endogenous lacZ under the
control of lacZpo (Fig. 6, lane 1) was insignificant.
chromosome of LE392 ( Fig. 6). All inserted copies
proved functional ( Table 1).
3.5. Conclusions
(1) We developed a simple and effective procedure for
construction of E. coli strains with multiple chromosomal insertions of foreign DNA. The system uses
elements of the Tn1545 site-specific recombination
module for random integration of the gene of
interest and drug resistance marker into the chromosome and the excision system of phage l for removal
of the drug marker upon integration of each copy.
(2) The presence of a thermoinducible chromosomal
copy of int-Tn in the host strain for integration
allows one to avoid recombination of the foreign
DNA fragment with an intracellular helper plasmid.
(3) The use of an excisable selectable cassette allows
easy identification of integrated forms. The drug
marker can be excised from the chromosome after
integration of each copy, which makes possible the
use of a single integrative plasmid to produce
multiple insertions and allows construction of such
strains without multiple drug resistance.
Acknowledgement
This work was supported by the grant C-1268 from
the Robert A. Welch foundation.
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