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Nucleic Acids Research Pages 1296-1299  

Gene replacement with linear DNA in electroporated wild-type Escherichia coli
Introduction
Materials And Methods
   Bacterial strains and plasmids
   Media
   Gene replacement with a plasmid target
   Gene replacement with a chromosomal target
   Assay for ATP-dependent double-strand DNA degradation
   Bacteriophage T4 and T4 gene2-in vivo test for exonuclease activity
Results
   Electrotransformation allows efficient gene replacement on a plasmid target
   Gene replacement on a chromosomal target by electrotransformation
   The exonuclease activity of RecBCD is reduced after electroporation
Discussion
Acknowledgements
References

Gene replacement with linear DNA in electroporated wild-type Escherichia coli

Gene replacement with linear DNA in electroporated wild-type Escherichia coli

Meriem El Karoui, Susan K. Amundsen1, Patrick Dabert+ and Alexandra Gruss*

Génétique Appliquée-URLGA, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas, France and 1Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, PO Box 19024, A1-162, Seattle, WA 98109, USA

Received November 12, 1998; Revised and Accepted January 12, 1999

ABSTRACT

Gene replacement using linear double-stranded DNA fragments in wild-type Escherichia coli transformation is generally inefficient due to exonucleolytic degradation of incoming DNA. Recombination-proficient strains, in which the exonucleolytic activity of RecBCD is inactivated, have been used as transformation recipients to overcome this difficulty. Here we report that gene replacements using linear double-stranded donor DNA can be achieved in wild-type E.coli if electrocompetent cells are used. Using a plasmid target, we obtained 102-103 gene replacement events/µg linear DNA. Using an independent chromosomal target, ~60 gene replacement events/µg linear DNA were obtained. The presence of Chi sites on the linear DNA, which are known to block DNA degradation and stimulate recombination in E.coli, had no effect on gene replacement efficiency in either case. RecBCD-mediated exonucleolytic activity was found to be diminished in electroporated cells. Electrotransformation thus provides a simple way to perform gene replacements in many E.coli strains.

INTRODUCTION

Gene targeting using linear double-stranded (ds)DNA fragments in wild-type Escherichia coli transformation is generally inefficient due to exonucleolytic degradation of incoming DNA. Recom-bination-proficient strains in which the exonucleolytic activity of RecBCD is inactivated (such as recD, recB recC sbcA and recB recC sbcB sbcCD mutants or strains which express bacteriophage [lambda] recombination functions) have been used as transformation recipients to overcome this difficulty (1-4). Recently, an approach was developed to obtain gene replacement in wild-type cells, in which the transforming linear DNA contained Chi sequences (5[prime]-GCTGGTGG-3[prime]) at both ends flanking the homologies (3). These sequences are known to attenuate RecBCD exonuclease activity and stimulate its recombination activity (5-7). Here we report that gene replacements using linear DNA without Chi sequences can be achieved in wild-type E.coli, on a plasmid as well as a chromosomal target, if electrocompetent cells are used. Electrotransformation seems to reduce the exonucleolytic activity of RecBCD in E.coli, thus allowing gene replacement to occur. This method provides a simple way to perform gene replacement in many E.coli strains.

MATERIALS AND METHODS

Bacterial strains and plasmids

Strains and plasmids used in these experiments are described in Tables 1 and 1.

Media

LB broth and agar plates, TB, BBL agar plates, minimal medium and phage suspension medium (SM) have been described (8). Ampicillin (Amp) was used at 100 µg/ml, kanamycin (Km) at 35 µg/ml and chloramphenicol (Cm) at 15 µg/ml.

Gene replacement with a plasmid target

The plasmid target (named p[Delta]Bla) is a pBR322 derivative with a 111 bp deletion in the [beta]-lactamase gene (bla). The intact bla gene is restored via a double exchange event with a linear DNA fragment (Fig. 1; see 9 for details of construction). In brief, primers were designed to PCR amplify a bla gene internal fragment covering the DNA deleted from p[Delta]Bla, plus an additional 360 bp flanking homology with bla. One primer couple contained double Chi sites while the other did not (9). To avoid having Chi sites at extremities (since recognition of Chi may require a minimal distance from the end), we added heterologous DNA at the ends of the linear fragment. For this purpose, the PCR fragments were cloned into a pBS derivative containing a Km-resistant (KmR) marker (kindly provided by P. Renault, INRA, Jouy en Josas, France); the final linear fragment containing DNA internal to the bla gene flanked or not by Chi sites and surrounded by heterologous DNA was excised on a PvuII fragment and its structure was confirmed by sequencing. Electrocompetent cells of strain TG1 carrying p[Delta]Bla were prepared and electrotransformed (resistance used 250 [Omega]) with the linear DNA as described (10). Cells were incubated for 90 min after electrotransformation and colony counts were performed after a 2 day incubation. Linear DNA samples were quantitated on ethidium bromide-stained agarose gels using marker DNAs of known quantities. Electrocompetence was determined by transforming cells with known amounts of supercoiled pACYC184 DNA and selecting for Cm resistance.

Table 1. Strain list
Strain Genotype Source/reference
TG1 (F[prime] traD36 LacIq [Delta](lacZ)M15) proA+B+/ supE [Delta](hsdM-mcrB)5(rk-mk-McrB-) thi [Delta](lac-proAB)  
V66 argA21 recF143 hisG4 met rpsL31 galK2 xyl-5 rac- [lambda]- F- (15)
V1904 as V66 but his+ (3)
AC113 [Delta](argA-thyA)232 IN(rrnD-rrnE)1 [lambda]- F- (16)
JC9387 thr-1 leu-6 thi-1 lacY1 galK2 ara-14 xyl-5 proA2 hisG4 argE3 rpsL31 tsx-33 mtl-1 recB21 recC22 sbcB [lambda]- F- (15)

Table 2. Plasmid list
Plasmid Description Source/reference
p[Delta]Bla pBR322 derivative with an internal deletion (ScaI-PvuI) in the bla gene (9)
pDA15 pBR322 derivative containing the his::kan insertion without Chi sites (3)
pDA16 as pDA15 with Chi sites on both ends of the his::kan insertion (3)
pDWS2 pBR322 derivative containing cloned recBCD genes of E.coli (17)

Gene replacement with a chromosomal target


Figure 1. Gene replacement strategy (plasmid target). The gene replacement target is plasmid p[Delta]Bla, which bears an internal deletion of bla ([Delta]bla). Linear transforming DNA contains an internal fragment of bla (blaint, black rectangle) which spans the bla deletion and has an additional 360 bp flanking homology with bla (gray rectangles). For the fragment Chi+, double Chi sites (shown as [chi][chi] in parentheses) are present adjacent to the homologous region. Wavy lines represent heterologous dsDNA tails. Double exchange homologous recombination would be required to convert cells to AmpR (bla+). Hatched rectangles on p[Delta]Bla represent bla DNA outside homologous regions (the figure is as in ref. 9; with permission from the National Academy of Sciences USA, © 1998).

The chromosomal target is the E.coli histidine synthesis (his) operon. Gene replacement results in the interruption of this operon by a KmR determinant. The construction of linear DNA used for targeting is as described (Fig. 22; see 3 for details). In brief, two pBR322 derivatives were constructed with a 3 kb fragment (hisGDC[prime]) of the his operon interrupted approximately in the middle by a KmR determinant (his::kan) with or without Chi sites flanking the his fragment. These plasmids (pDA15 and pDA16) were then linearized by EcoRI digestion and the 6.5 kb fragment was purified (3). Electrocompetent cells of V1904 were prepared and electrotransformed (resistance used 250 [Omega]) with the linear DNA fragments as described (11). Cells were incubated for 1 h after electrotransformation and colony counts were performed after 24 h incubation. KmR colonies were patched on fresh LB-Km plates and replicated onto minimal medium lacking histidine and onto LB-Amp plates. Gene replacement events were scored as KmR, His-, AmpS transformants. Linear DNA samples were quantitated by determining the UV absorption spectrum from 220 to 340 nm using a Shimadzu UV-1201 spectrophotometer. Electrocompetence was determined by transforming cells with known amounts of supercoiled pDA15 or pDA16 DNA, selecting for Km resistance.


Figure 2. Gene replacement strategy (chromosomal target). The gene replacement target is the his operon on the E.coli chromosome. Linear transforming DNA contains hisGDC’(grey rectangle), interrupted by the KmR gene (KmR, black line). For the Chi+ fragment, single Chi sites (shown as [chi] in parentheses) are present adjacent to the homologous regions. Wavy lines represent the chromosome. Double exchange homologous recombination produces KmR His- AmpS cells (3).

Assay for ATP-dependent double-strand DNA degradation

An overproducing RecBCD strain (AC113 carrying the plasmid pDWS2; 17) was prepared for electroporation using standard procedures (10) and aliquots of ~5 × 109 cells were frozen. Electroporation was performed in triplicate on thawed aliquots using varying resistance (0, 200, 600 or 800 [Omega]). Following electroporation, 1 ml of LB was added to each sample and the cells incubated at 37°C for 10 min. The cells from each electroporation condition were pooled, pelleted and crude extracts prepared as described previously (12). Extracts were assayed for ATP-dependent DNA solubilization of 3H-labeled phage T7 dsDNA as described (8).

Table 3. Gene replacement frequencies in electrocompetent E.coli host using linear donor DNA and plasmid or chromosomal target
Strain Supercoiled DNA
Transformants/µg DNA		 Linear DNA
Plasmid gene
replacements/µg DNAa		 Chromosomal gene
replacements/µg DNAb
Chi0 c Chi+ Chi0 Chi+
TG1 (p[Delta]Bla) 3 × 109 1012 482 - -
V1904 5 × 108 - - 60 65
, not done.
aExperiments were performed with 2, 6, 12, 50, 100 or 500 ng of linear DNA. The number of transformants is proportional to the amount of DNA used within these concentrations and values shown are extrapolated to 1 µg. In total, 670 ng of either Chi+ or Chi0 DNA was used.
bExperiments were performed with 100 ng of linear DNA. Values shown are extrapolated to 1 µg. Results are means of five experiments on 3 days for the Chi0 fragment and two experiments for the Chi+ fragment.
cChi0 indicates DNA has no Chi sites. Chi+ indicates DNA fragment contains a single or double Chi site near each end(see Figs 1 and 2 and Materials and Methods).

Bacteriophage T4 and T4 gene2-in vivo test for exonuclease activity

Strain V66 (recBCD+) was electroporated at 0, 200 or 600 [Omega] as described (see above). After electroporation 1 ml of TB was added and cells were incubated at 37°C for 20 min. Aliquots of 5 × 108 cells were mixed with 2.5 × 107 particles of T4 or T4 gene2- phage (as assayed on strain JC9387; recBC) and incubated at 37°C for 10 min. The bacteria-phage mixtures were serially diluted in SM and 0.1 ml of the dilution added to 0.2 ml of E.coli strain JC9387 as indicator bacteria. To this, 2.5 ml of soft top agar was added and the mixture was poured onto BBL plates. After overnight incubation at 37°C the number of plaque-forming units was determined.

RESULTS

Electrotransformation allows efficient gene replacement on a plasmid target

We designed a model system to examine gene replacement on a plasmid target using linear DNA in transformed electrocompetent wild-type E.coli. The gene replacement plasmid target is an internally deleted [beta]-lactamase gene (bla) which is present on p[Delta]Bla, a pBR322 derivative (9; Fig. 1; Materials and Methods). Restoration of bla on p[Delta]Bla by gene replacement with a linear molecule requires a double exchange event (Fig. 1). The linear DNA fragments used contain the internal region missing from p[Delta]Bla plus ~360 bp of adjacent bla DNA (Materials and Methods). Cells which have undergone gene replacement are selected as AmpR. As Chi is known to attenuate RecBCD exonuclease activity and stimulate its recombination activity (5-7) we tested linear fragments with no Chi sites (referred as Chi0) as well as identical fragments containing Chi sites (referred as Chi+). On the Chi0 fragment, heterologous DNA flanked the regions of homology; on the Chi+ fragment, double Chi sites flanked the homology on either side, followed by the same heterologous DNA (Fig. 1). Note that no Chi sites were present on the linear DNA fragments other than those added in Chi+ fragment. To determine the efficiency of gene replacement, we transformed electrocompetent TG1 cells containing p[Delta]Bla with the linear DNA fragments and counted the number of AmpR transformants obtained with each DNA (Table 3).

For both fragments Chi+ and Chi0, 102-103 gene replacement events were obtained per µg linear DNA and we observed no significant difference in the number of gene replacement events using either fragment within a single experiment. AmpR transformants restored the plasmid-carried bla gene, as confirmed by PCR (data not shown). Electrotransformation using PCR-amplified linear DNA fragments (rather than plasmid-derived linear DNA) gave similar results (data not shown). Thus, efficient gene replacement was obtained by electrotransformation with linear DNA fragments and a plasmid target. The efficiency was not altered by the presence of Chi sites.

Gene replacement on a chromosomal target by electrotransformation

To test whether E.coli electrotransformation also allows efficient gene replacement on a chromosomal target, we made use of a second model system in which the target was the chromosomal his operon. To generate the linear DNA fragment, a pBR322 derivative, containing the his::kan fragment (a 3 kb segment of the his operon interrupted by a KmR determinant), was linearized by EcoRI restriction (Materials and Methods; 3). Homologous gene replacement of the chromosomal his locus with this fragment results in His- KmR cells. The AmpR determinant of pBR322 is lost during gene replacement (3). The linear DNA fragments Chi+ and Chi0 were designed such that single Chi sites or no Chi flanked the hisG and hisC[prime] genes (Fig. 22). Note that no Chi sites were present on the linear DNA fragments other than those added in Chi+ fragment. To determine the efficiency of gene replacement, we transformed electrocompetent V1904 cells with the linear DNA fragments and counted the number of KmR transformants which were His- and AmpS (Materials and Methods; Table 3). For both Chi+ and Chi0 fragments ~60 gene replacement events were obtained per µg linear DNA. These results show that gene replacement on a chromosomal target can be obtained by electrotransformation. The efficiency is not altered by the presence of Chi sites.

Table 4. Exonuclease activity of RecBCD is reduced after electroporation
Resistance used for
electroporation ([Omega])
AC113(pDWS2)b		 ATP-dependent dsDNA
exonuclease activity (U/mg protein)		 Phage forming an
infection centera (%)
V66c   T4 gene2- T4
0 700 50 <4 73
200 40 7 11 79
600 12 5 58 64
800 <3 <3 - -
-, not done.
aThe strain used is V66. Results are means of two experiments. Total phage titers were determined on strain JC9387.
bStrain contains cloned recBCD genes. Results are means of two experiments; individual values differ by <10%.
cResults are from one experiment.

The exonuclease activity of RecBCD is reduced after electroporation

Our results show that the frequencies of gene replacements with linear DNA are not affected by the presence of Chi sequences on the linear fragments. This could be due to an inactivation of RecBCD nucleolytic activity during electroporation. After electroporation we measured the ATP-dependent dsDNA exonuclease activity in crude extracts of a strain overproducing RecBCD enzyme (AC113, containing the plasmid pDWS2, with the cloned recBCD genes). We observed a dramatic decrease in in vitro exonuclease activity when cells were electroporated at 200, 400 and 600 [Omega] (cells are electroporated at 250 [Omega] in routine electrotransformation protocols) (Table 4). Comparable results were obtained in an E.coli strain containing the chromosomal copy of RecBCD (V66). This result was confirmed in vivo by examining sensitivity to bacteriophage T4 gene2- infection. Bacteriophage T4 gene2- DNA is sensitive to exonuclease degradation (13) and its plaque-forming ability provides a simple test to evaluate host nuclease activity (14). A wild-type strain, which is normally resistant to bacteriophage T4 gene2- infection, became very sensitive upon electroporation. The infection capacity of the T4+ bacteriophage, which is not sensitive to exonuclease degradation, was not altered by electroporation. Taken together, these results show that the exonuclease activity of RecBCD is diminished after electroporation.

DISCUSSION

Our results show that gene replacement events in wild-type E.coli can be readily selected using linear donor DNA when introduced into electrocompetent cells. This could be due to partial inactivation of RecBCD exonuclease activity: reduced degradation of the linear DNA fragment could allow the gene replacement event to occur.

Two other approaches have been recently developed to obtain gene replacement with linear DNA. The first method uses the property of Chi sites to regulate RecBCD exonuclease activity and stimulate recombination. Chi sites present near the ends of linear DNA fragments stimulate the frequency of gene replacement events when wild-type E.coli cells are made competent by treatment with CaCl2 (3). One drawback of this method is that it requires DNA constructions that add Chi sites at the fragment extremities. The second method uses the bacteriophage [lambda] recombination functions to stimulate gene replacement (4). Although extremely efficient, this system requires the use of a particular E.coli strain and thus limits its range of use.

In contrast, the method described here to obtain gene replacement can be used in many different E.coli strains and does not necessitate special DNA constructions. The frequencies of gene replacement events obtained (with a chromosomal target) are comparable to those obtained in the Chi-stimulated recom-bination method (3). Electrotransformation may thus constitute a straightforward method to obtain gene replacements with linear DNA in wild-type E.coli on plasmid and chromosomal targets. It may also be used to make gene disruptions on plasmid-carried targets which can then be transferred to the organism of interest.

ACKNOWLEDGEMENTS

We thank Sophie Sourice for discussions and her valuable assistance in DNA sequencing. We are grateful to Gerry Smith for his interest in this work. This work was supported, in part, by grant ALTF 622-1992 from the European Molecular Biology Organization to P.D. and research grant GM31693 from the National Institutes of Health to G. R. Smith.

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*To whom correspondence should be addressed. Tel: +33 1 34 65 21 68; Fax: +33 1 34 65 20 65; Email: gruss@biotec.jouy.inra.fr
+Present address: Laboratoire de Biotechnologie de l’Environnement, Institut National de la Recherche Agronomique, Avenue des Etangs, 11100 Narbonne, France

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