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The availability of complete bacterial genome sequences has provided a wealth of information on the molecular structure and organization of a myriad of genes and ORFs whose functions are poorly understood. A systematic mutational analysis of genes in their normal location can provide significant insight into their function. Using recombinant DNA techniques it has become possible to create mutations in vitro, which subsequently may be introduced into E. coli chromosome.
There are several methods to introduce a mutant sequence synthesized in vitro into the E. coli chromosome. The two main previously established methods are the 'in-out' method and the 'linear fragment' method. In the 'in-out' method, [3, 4] a mutant sequence is introduced into the cell on a multicopy circular plasmid. A Rec mediated single homologous crossover results in cointegration of the whole circle into the genome at the target site with the plasmid vector between a wild type and mutant copy of the target sequence ('in'). The cointegrate is resolved by a second single crossover ('out'). When in and out crossovers span the mutant site, the desired mutation is transferred to the genome. The 'linear fragment method' [2, 5], utilizes the Red recombination system encoded by bacteriophage lambda genes gam, bet and exo which operates on linear DNA. Electroporation is used to introduce a linear DNA fragment carrying the synthesized mutation directly into the cell where Red favors double crossover events in the progeny since a single crossover would result in a chromosome break. Incorporation of the mutation into the chromosome occurs where the double crossover spans the mutant site.
Here we present the 'in-out' gene replacement method. Phenotypic screens, Southern analyses or PCR analysis are then used to identify the replaced allele. The key to this procedure involves the use of vectors that cannot replicate under conditions used for selection of the cointegrate. Examples of such vectors for E.coli include ColE1-derived plasmids, which do not replicate in polA mutants  a temperature-sensitive pSC101 replicon , and a phagemid-based vector . In this work we describe the construction of E. coli strain with aroA CP4 deletion mutant, and construction of a vector that appears to express aroA CP4 deletant gene (aroAâˆ†50). We used pMAK705 vector (5.6kb), which has a temperature-sensitive origin of replication providing resistance to chloramphenicol. This allowed selection of cells with a plasmid integrated into the host chromosome, using chloramphenicol resistance at the non-permissive temperature of 44°C.
Materials and methods
1. Bacterial strains and plasmids: The strains used in this study are E. coli wt12 - gyrA96, ApR, TcR, RifR, EryR , MC4100 - araD139, âˆ†(argF lac)U169, rpsL150, relA1, flbB5301, deoC1, ptsF25, rbsR , ER2799-âˆ†aroA, gln-  were used in all our experiments. The plasmids used were pMAK705 , pKKâˆ†50-Cm - deletion mutant in aroA CP4 gene in expression vector pKK233-2 (deletion 52bp), CmR, and pMAKâˆ†50 (this study).
2. DNA techniques: Restriction endonucleases HindIII and BamHI, T4 DNA ligase (Gene Craft, Germany) and PCR kit (Promega) were used according to the specifications of the manufacturer. E. coli chromosomal DNA and Plasmid DNA was isolated by Qiagen kit. Isolation of restriction fragments for subsequent cloning was accomplished by elution from 1% agarose gel (SBS agarose) and purified by Qiagen gel extraction kit.
3. pMAKâˆ†50 vector construction: pKKâˆ†50 plasmid was digested with HindII and BamHI restriction enzymes resulting in aroAâˆ†50 fragment (Fig. 2a). This fragment cloned in the HindIII-BamHI site of polylinker of pMAK705 resulting in the recombinant vector pMAKâˆ†50 (Fig. 2b).
4. Electroporation: Electro-competent E. coli cells were obtained by washing exponentially growing cells three times with ice-cold distilled H2O and concentrating 300-fold with 10% glycerol. Electroporation was carried out in 0.2-cm electroporation cuvettes using a BioRad GenePulser II set to the following parameters: 200 Ω, 25 µF and 2.5 kV. Shocked cells were diluted 3-fold in LB, incubated for 1 h at 30°C with aeration and subsequently plated on appropriate media.
5. Gene replacement: pMAK705 plasmids carrying a mutant allele was delivered into the target cell by electroporation. Cells were spread on LB plates supplemented with an appropriate antibiotic (LBAb) and incubated at 28°C overnight. Several colonies were then picked and restreaked on LBAb plates and incubated at 44°C overnight. Typically many large colonies are formed over the background of small colonies. These large colonies carry the plasmid inserted into the chromosome; however, at this point usually the colonies contain cells harboring unintegrated plasmids as well. To obtain cells that are cured of the free plasmid, a few large colonies are picked individually, restreaked on LBAb plates and grown at 28°C overnight. This cycle is performed for three rounds. Colonies should be uniform in size this time, and finally the site of insertion can be verified by PCR using an appropriate primer pair.
6. Isolation of plasmid-chromosome cointegrates: The aroA CP4 deletion mutant was cloned into pMAK705 which has temperature sensitive replicon. The plasmid replicates at 28°C but not at 44°C. We used 44°C as the nonpermissive temperature for temperature sensitive mutations. The transformed cells from 28°C were inoculated onto prewarmed chloromphenicol plates at 44°C. This cycle is performed for three rounds.
7. Identification of resolution products and chromosomal integration target gene: When the plasmid is integrated into the chromosome, replication from the plasmid origin is deleterious to the cell. Accordingly, when the cointegrates identified at 44°C were subsequently grown at 28°C, the permissive temperature for replication of the plasmid, a second recombination event occurred, regenerating free plasmid in the cell. Individual colonies from the 44°C plates were inoculated onto plates containing chloromphenicol and grown at 28°C overnight. Three more such cycles were carried out. Finally the colonies that were grown at 44°C no longer had plasmid DNA integrated into the chromosome. At last the chromosomal DNA was isolated from the cells grown at 28°C. PCR amplification of aroAâˆ†50 was carried out to find out whether the aroAâˆ†50 being integrated into the chromosomal DNA using the primers:
For2 (GACGCCATGGCTCACGGGTGCAAGCAGCCGTCC) and
Results and discussion
Temperature-sensitive mutants of pSC101 have proven to be valuable tools for genetic manipulation in E. coli. For example, thermosensitive pSC101 derivatives have been used for gene disruption and allelic exchange with the bacterial chromosome . Despite the proven value of thermosensitive pSC101-derivative vectors most were constructed for specialized uses and are not well suited for use as cloning vectors. In general, these plasmids lack many characteristics usually associated with versatile cloning vectors such as the availability of a variety of unique restriction enzyme recognition sites, a convenient means of screening for recombinant plasmids, and the option to use different antibiotic selections .
and Koshland, 1983); Fig 1b: pMAKâˆ†50 vector constructed by cloning aroA CP4 deletion mutant in pMAK705.
In our studies we used pMAK705 plasmid which is thermosensitive and cloning in this was cumbersome. A number of genetic techniques require a plasmid vector with an origin of replication (ori) that is conditionally active; i.e., the plasmid fails to replicate under nonpermissive growth conditions. Growth under nonpermissive conditions results in plasmid elimination or chromosomal integration at regions of significant DNA homology. To confirm that the chloromphenicol deletion-insertion construct of the aroA gene had been transferred to the chromosome, chromosomal DNA was isolated from the strains and subjected to PCR analysis with proper primers to amplify the mutant target gene.
Construction of a bacterial mutant by using recombinant plasmid sequences altered in vitro involves three steps (Fig 1): (i) integration of a plasmid into the host chromosome via recombination; (ii) resolution of the plasmid from the host chromosome, leaving the altered sequence in the chromosome but removing the wild-type gene; and (iii) detection of the resulting mutant. In the first step, a plasmid carrying the altered sequence and the genes for chloromphenicol resistance is introduced into E. coli wt-12 and ER2799 strains. Since the plasmid cannot replicate in the cytoplasm at 44°C, chloromphenicol-resistant transformants will have plasmid integrated into the chromosome. In the second step, a plasmid integrate is grown at 28°C the permissive temperature for replication of the plasmid, a second recombination occurred, regenerating free plasmid in the cell. In the third step, screening for the new mutant can be carried out either phenotypically or by characterization of the chromosomal DNA using Southern hybridization analysis. In our case the mutant gene integrated into chromosome is being amplified by using the appropriate primers.
In this study we have described construction of E. coli strain by recombination between the chromosome and the plasmid carrying a mutant gene (aroAâˆ†50) and the plasmid segregation allowing the simple and efficient isolation of mutants via a selection for the aroAâˆ†50 in the (now chromosomal) mutant gene. We have used a thermosensitive integration vector pMAK705 to facilitate the gene replacement. E. coli mutants were constructed carrying a aroAâˆ†50 CP4 gene using phenotypic selection. This strain with chromosomal integrated aroA deletant will be used for the study of horizontal gene transfer (HGT) in the future work.
The work was supported by grant of Slovak Grant Agency APVT-20-17102.
 Chen, L., Pradhan S., Pradhan, S., Evans Jr. T. C. (2001): Gene 263: p. 39-48.
 Datsenko, K.A., Wanner, B.L., (2000): Proc. Natl. Acad. Sci. USA 97: p. 6640-6645.
 Hamilton, C. M., M. Aldea, B. K. Washburn, P. Babitzke, and S. R. Kushner. (1989):
J. Bacteriol. 171: p. 4617-4622.
 Link, A.J., Phillips, D., Church, G.M., (1997): J. Bacteriol. 179: p. 6228-6237.
 Murphy K.C., Campellone K.G., Poteete A.R. (2000): Gene 246: p. 321-330.
 Phillips, G. J., (1999): Plasmid 41, 78-81.
 Renczésová, V. (2005): Diploma work, PriFUK Bratislava.
 Saarilahti, H. T., and E. T. Palva. (1985): FEMS Microbiol. Lett. 26: p. 27-33.
 Slater, S., and R. Maurer. (1993): J. Bacteriol. 175: p. 4260-4262.