- Methodology article
- Open Access
pBAM1: an all-synthetic genetic tool for analysis and construction of complex bacterial phenotypes
© Martínez-García et al; licensee BioMed Central Ltd. 2011
- Received: 20 November 2010
- Accepted: 22 February 2011
- Published: 22 February 2011
Since publication in 1977 of plasmid pBR322, many breakthroughs in Biology have depended on increasingly sophisticated vector platforms for analysis and engineering of given bacterial strains. Although restriction sites impose a certain format in the procedures for assembling cloned genes, every attempt thus far to standardize vector architecture and nomenclature has ended up in failure. While this state of affairs may still be tolerable for traditional one-at-a-time studies of single genes, the onset of systems and synthetic biology calls for a simplification -along with an optimization- of the currently unwieldy pool of genetic tools.
The functional DNA sequences present in the natural bacterial transposon Tn5 have been methodically edited and refactored for the production of a multi-purpose genetic tool named pBAM1, which allows a range of manipulations in the genome of Gram-negative bacteria. This all-synthetic construct enhances the power of mini-transposon vectors for either de-construction or re-construction of phenotypes á la carte by incorporating features inspired in systems engineering: modularity, re-usability, minimization, and compatibility with other genetic tools. pBAM1 bears an streamlined, restriction site-freed and narrow-host range replication frame bearing the sequences of R6K oriV, oriT and an ampicillin resistance marker. These go along with a business module that contains a host-independent and hyperactive transposition platform for in vivo or in vitro insertion of desired DNA into the genome of the target bacterium. All functional sequences were standardized for a straightforward replacement by equivalent counterparts, if required. pBAM1 can be delivered into recipient cells by either mating or electroporation, producing transposon insertion frequencies of 1.8 × 10-3 and 1.02 × 10-7, respectively in the soil bacterium Pseudomonas putida. Analyses of the resulting clones revealed a 100% of unique transposition events and virtually no-cointegration of the donor plasmid within the target genome.
This work reports the design and performance of an all-synthetic mini-transposon vector. The power of the new system for both identification of new functions or for the construction of desired phenotypes is shown in a genetic survey of hyper-expressed proteins and regulatory elements that influence the expression of the σ54-dependent Pu promoter of P. putida.
- Transposition Event
- Broad Host Range Plasmid
- MAD1 Strain
- tnpA Gene
The issue of modularity in genetic constructs has been present in the microbiological literature since the onset of recombinant DNA . Despite various attempts to format vector structure and nomenclature , there is not yet any generally accepted standard for plasmid architecture or physical assembly of cloned DNA sequences. This state of affairs is rapidly becoming a bottleneck as we move from handling just a few genes in typical laboratory organisms into analysing and massively refactoring the genomes of very diverse bacteria. The notion of formatted genetic tools for the analysis and stable engineering of microorganisms was pursued in the early 90s (among others) with the design of the so-called mini-transposon vectors . These allowed stable insertions of foreign DNA into the chromosome of virtually any Gram-negative target. Tn5-derived constructs presented a large number of advantages over their plasmid-based counterparts for introduction of transgenes into many types of bacteria [3–5]. These included maintenance without antibiotic selection, long-term stability and re-usability for generating multiple insertions in the same cells, with no apparent size limits. Yet, the original design of such mini-transposons [4, 5] was plagued with problems, such as the inheritance of long, non-functional DNA fragments carried along by the intricate cloning-and-pasting DNA methods of the time. These were also afflicted by the excessive and inconvenient number of non-useful restriction sites scattered along the vectors, and the suboptimal transposition machinery encoded in them. Despite downsides, the mini-transposon-bearing pUT plasmid series  are still to this day one of the most popular vector platforms for analysis and engineering of Gram-negative bacteria. In fact, every successful feature of the classical mini-Tn5s and its delivery system is originated in mobile elements (broad host range plasmids and transposons), which are naturally evolved to thrive in a large variety of hosts. In particular, the Tn5 transposition system requires exclusively the transposase encoded by tnpA, and the terminal ends of the transposon as the substrate. This affords transposition in a fashion virtually independent of the host, thereby qualifying as an orthogonal biological machinery that expands the utility of the vectors to virtually any host .
In this work we have exploited the current ease of DNA synthesis for a dramatic remake of the original mini-Tn5 transposon vector concept. In this process, the most attractive features have been enhanced while each of the drawbacks (identified along 20 years of use in hundreds of laboratories) has been eliminated. To this end, we have revisited the functional modules that shape the vector and have edited the corresponding DNA sequences to minimize them, improve their functionality and make them entirely modular and exchangeable. The final product was the entirely synthetic construct that we have named pBAM1 (for born-again mini-transposon), which multiplies the benefits of the earlier designs. We show below that this genetic tool is most advantageous not only for random mutagenesis studies on a target bacterium such as Pseudomonas putida, but also for implantation of functional cargos into its genome, be they one (or few) transgene(s), a transcriptional reporter, or a complex genetic or metabolic circuit. The applications are illustrated below in two different contexts. One regards the identification of new functions that influence the regulation of the catabolic σ54-dependent Pu promoter of P. putida. The other involves the visualization of the intracellular targeting of highly expressed proteins in individual bacteria by means of random generation of GFP protein fusions.
Rationale of the pBAM1 layout and editing of its functional modules
The first key feature of pBAM1 is the utilization of the narrow host-range origin of replication of plasmid R6K as the vegetative oriV of the construct for its proliferation. This origin is strictly dependent on the so-called π protein (encoded by the pir gene of R6K). The oriV and the pir gene of R6K can be separated and made to function in trans . This makes replication of any covalently close circular (ccc) DNA bearing such an oriV entirely dependent on the provision of the p protein, either from a second plasmid or from the chromosome. This feature has been exploited for the development of a number of conditional systems that make replication of a given construct addicted to host strains of E. coli that express the pir gene . Virtually all of such existing systems carry the R6KoriV-containing 420 bp fragment from pGP704 plasmid . This naturally occurring DNA sequence is not only longer than necessary for the oriV function, but it also carries an internal HindIII site in the midst of the repeats that are recognized by the replication machinery [9, 10]. Moreover, this segment in pGP704 has flanking EcoRI and BamHI sequences that prevent the cognate restriction enzymes being used for cloning. For pBAM1, the whole oriV region was streamlined to a minimum (392 bp) and the internal HindIII removed (while keeping a sequence in the former site with similarity to the functional repeats). Finally, the termini of the segment were furnished by the infrequent restriction site AscI to create the origin of replication module. These changes did not affect any of the properties described for the natural R6KoriV sequences . pBAM1 and its derivatives are maintained in the specialized strain E. coli CC118λpir, which expresses the π protein from a lysogenic phage .
The next module of the plasmid frame was the sequence that contains the origin of transfer oriT (Figure 1) and enables transfer of pBAM1 from the host strain to a new recipient, when recognized by the conjugative machinery encoded by the broad host range plasmid RK2, also called RP4 . Since the RP4/RK2 conjugal transfer system is the most promiscuous of all DNA mobilization device known, the presence of oriT allows mobilization of pBAM1 into virtually any Gram-negative or Gram-positive bacteria  and can even be passed into fungi  and eukaryotic cells , provided that the construct is exposed to the action of the Tra proteins of RP4 . This transfer can be made by either setting up a tri-parental mating mixture with a donor strain (e.g. E. coli CC118λpir) bearing the R6KoriV/RP4oriT plasmid, a recipient bacterium and helper cells bearing the mob/tra region of RP4 cloned in a plasmid which does not replicate in the recipient . As an alternative, the donor λpir+ strain may have the tra/mob functions integrated in its chromosome (for instance, E. coli S17-1λpir) allowing bi-parental mating . Other λpir+E. coli donor strains such as E. coli RH03, which have been engineered to facilitate counter-selection, are also eligible to this end . The oriT region employed in most plasmid vectors designed for mobilization purposes is exceedingly large (1728 bp) and flanked by BamHI sites . As before, we trimmed down the oriT to the minimum of 244 bp required for functionality , eradicated one SfiI site present within the core oriT sequence (to allow its inclusion in the polylinker of the vector) and the streamlined module was flanked by the two rare enzyme sites FseI and PshAI. Note, however, that in some cases the plasmid can just be electroporated into target cells and conjugation may not be necessary, although the efficiency is considerably lower. Since the plasmid transferred to the recipient by conjugation or electroporation cannot in any case replicate because of the lack of the p protein, this process is called suicide delivery.
The last module of the plasmid frame of pBAM1 was the bla gene that encodes a β-lactamase, endowing Ap resistance as selective marker. We kept the natural P3 promoter of the natural bla gene to control its expression;  and maintained the protein sequence of the enzyme that is employed by many other vectors , but the codon usage of the gene was optimized for E. coli and potentially conflicting restriction sites removed. Furthermore, transcriptional terminators from the trpA gene (alpha subunit of the tryptophan synthase from E. coli) and the gene VIII from phage fd were placed upstream and downstream of the bla gene, respectively, to avoid transcriptional readthrough from neighbouring sequences. Finally, this selection marker module was flanked by SwaI and PshAI sites, as shown in Figure 1.
Next come the elements engineered in pBAM1 for causing insertions of cloned DNA into the genome of the target strain. These include a segment encoding the transposase gene tnpA lying outside but adjacent to a DNA segment flanked by the terminal sequences of Tn5 (i.e. the mini-transposon itself). The Tn5 transposase recognizes both end-sequences and catalyzes the transfer of the mobile module from the donor replicon to the target genome, where it randomly inserts (there is a slight preference for G/C at both ends of the 9-bp target sequence; ). The configuration in pBAM1 exploits the fact that the Tn5-carried tnpA gene also works well when located outside the mobile element, although it still needs to be in cis in respect to the target sequences of its gene product [20, 21]. The sequence of the Tn5 tnpA gene of pBAM1 was edited to enhance a number of desirable characteristics. First, instead of the naturally occurring gene, which has evolved to mediate a very low level of transposition, we re-designed tnpA to endow its product with hyperactivity . This included an E54K substitution, which increases transposase binding to the terminal OE sequence, a M56A change that blocks the synthesis of the Inh protein (a trans-dominant negative truncation of TnpA that represses transposition), and a L372P replacement that enhances TnpA dimerization, thereby improving its activity . As before, to eliminate inconvenient restriction sites, the NotI sequence indigenous to the IS50R part of Tn5 was removed by a silent substitution G504->C . In addition, the tnpA stop codon TGA was changed by the more efficient TAA termination codon. Otherwise, the edited transposase gene was expressed through its natural T1 promoter. However, as tnpA expression is downregulated by methylation, the two dam recognition sites (5'-GATC-GATC-3') present within this promoter region were changed to 5'-AATC-GATG-3' as described . The sum of all these operations yielded an optimized transposase variant carried by a 1524 bp segment flanked by PmeI and SwaI sites.
Although a large number of useful sequences can be placed between ME-I and ME-O, the mini-transposon carried by pBAM1 carries a Km resistance gene (neo) from Tn903 as a default selection marker, as well as what we call a cargo site containing a polylinker for general cloning purposes. As before, the natural neo sequence (GenBank: V00359;  was edited to improve codon usage and to eliminate the naturally occurring SmaI and HindIII sites at positions 306 and 550 respectively from the start codon of the neo gene. The resistance gene was expressed through its natural, broad host range promoter, which spans 81 bp upstream of the start codon of the neo gene, the entire KmR cassette being bracketed by terminal AatII and SanDI restriction sites. These anchor the neo gene within the transposable segment of pBAM1 and allow its replacement when required by other selectable markers. The cargo site consists of a polylinker for thirteen unique restriction sites flanked with sites for the rare cutter enzyme sites SpeI and PacI. One site (NotI) is however repeated at both ends of the polylinker, because its internal deletion reconstructs a short NotI-SfiI sequence that makes it compatible with earlier versions of mini-transposons [4, 5]. In contrast to these, however, the cloning sites of the polylinker are unique in pBAM1, making unnecessary the two-step cloning protocols that afflicted the former chromosomal insertion strategies . The final assembly thus has the start codon of the neo gene 107 bp downstream of the ME-I, while the stop codon is 174 bp downstream of the ME-O, the total length of the optimized element becoming 1135 bp (Figure 2A).
The modular layout of the functional segments of pBAM1 allows the replacement of each of them by equivalent counterparts, leaving intact the others. We thus argue that the rare sites that punctuate the structure of the vector (Figure 1) provide a useful standard for physical assembly of equivalent systems with other origins of replication, other transposable systems e.g. mariner , Tn7 , and other selection markers. Once the study of each module was made along the lines mentioned above and the sequences edited in silico, the whole was assembled to produce a unique sequence of 4384 bp that was chemically synthesized.
Validation of pBAM1
To assess the functionality and versatility of the new synthetic vector we passed it through several experimental tests to check that the plasmid and the new minimized standard features worked as expected. First we verified that the construct was stably propagated in E. coli CC118λpir, as a medium-to-high copy number plasmid (not shown). This confirmed that the editing of the HindIII site in one of the repeats of R6KoriV previously believed to be critical for replication  was tolerated by the plasmid without any detrimental effect. We next tested two different methods for suicide delivery of the plasmid into a recipient strain (P. putida KT2440), which is a good representative of the non-enteric Gram-negative bacteria widely used in industrial and environmental microbiology [30–32]. First, we employed a standard tri-parental mating (see Materials and Methods) for verifying the transposition process and determining the optimum period of time required for constructing a saturated transposition insertion library. To this end, the mating mix was allowed to conjugate for 1 to 18 h on filters laid on LB plates. At the times indicated, the cells on the filters were resuspended and plated onto M9-citrate agar with Km for removal of the donors and selection of P. putida clones bearing insertions of the mini-Tn5 element. As shown in Additional File 1 (Figure S1), the average frequency of KmR exconjugants ranged from 0.006 ± 0.008 × 10-3 after one hour of mating, to 6.2 ± 0.15 × 10-3 at eighteen hours. The number of exconjugants at longer times, although higher, were not considered as they surely reflected the amplification of earlier transposition times through cellular division, instead of new transposition events. In view of the bimodal shape of the time course of Figure S1 (see Additional File 1) we picked 5 h as the most useful time for maximum conjugation/transposition events with a minimum of growth. The next step was to examine whether exconjugants had undergone authentic transposition events or they resulted from the cointegration of pBAM1 into the host genome. 200 colonies were randomly selected and their sensitivity to the plasmid marker (ApR) tested. All 200 KmR clones turned out to be sensitive to the β-lactam antibiotic ampicillin (500 μg ml-1), thereby indicating that the insertion of the mini-transposon carried by pBAM1 had occurred as expected.
Transposition frequencies of pBAM1
Analyses of exconjugants
1.8 ± 0.53 × 10-3
1.02 ± 0.38 × 10-7
Primers used in this study
Sequence 5' → 3'
PCR round 1
PCR round 2
PCR round 1
PCR round 2/sequencing
PCR round 1
PCR round 2/sequencing
PCR round 1
PCR round 2/sequencing
Exploration of the regulatory landscape of the catabolic Pu promoter of P. putida
The σ54-dependent Pu promoter employed above is the principal regulatory element at play in the regulation of a complex system for biodegradation of m-xylene in strain P. putida mt-2 . P. putida MAD1 strain keeps the essential components of the m-xylene sensor system, fused to a lacZ reporter. The high performance of pBAM1 just described was thus exploited to survey the genome of P. putida for genes which could influence -not abolish- lacZ output in the hope of identifying novel functions which may well shed some light on the physiological regulation of the Pu promoter . To this end, the collection of ~40.000 KmR colonies derived from P. putida MAD1 plated on M9-citrate with kanamycin and exposed to m-xylene was examined for the appearance of paler blue tones or unusual patterns of Xgal in the otherwise dark blue of the control colonies that peak at the colony centre. Seven of these (Figure 3D and Table S3 of Additional File 1) were chosen for further analysis. The sequence of the corresponding sites of insertion revealed at least two types of genes that influenced the outcome of the Pu-lacZ reporter. One group is constituted by an insertion in dnaJ, which appears to downregulate Pu (Figure 3D). DnaJ is a heat-shock protein that stimulates the ATPase activity of DnaK  and is perhaps involved in the pathway for proper folding of σ54 (RpoN; ). A similar Xgal distribution pattern is observed when the PP1841 gene is disrupted (Figure 3D). Yet, the most unusual phenotype of the Pu-lacZ fusion carried by P. putida MAD1 appeared in an insertion within the intergenic region between cstA, a gene, which encodes a carbon-stress response protein , and PP4642, a type IV pilus assembly gene. In these cases (Figure 3D), the colonies displayed a double-ring distribution of the dye that suggested an influence of either or both of these proteins in adjusting the physiological control of Pu activity . Other interesting phenotypes were produced by mutations in cysD and cysNC genes, the loss of which produce small, slow-growing colonies with a distinct fisheye distribution of Xgal. These mutations are expected to bring about a general deficiency of cysteine , which could directly or indirectly affect transcriptional activity (Additional File 1, Table S3). Needless to say, these are preliminary observations that require further examination (see other insertions in Table S3 of Additional File 1). In the meantime, these results illustrate the power of the genetic tool employed for tackling regulatory phenomena.
Survey and localization of highly-expressed proteins in Pseudomonas putida
Bacteria and plasmids
Δ(ara-leu), araD, ΔlacX174, galE, galK, phoA, thi1, rpsE, rpoB, argE (Am), recA1, lysogenic λpir
SmR, hsdR-M+, pro, leu, thi, recA
mt-2 derivative cured of the TOL plasmid pWW0
KT2440 RifR, TelR, xylR+, Pu-lacZ
CmR; oriColE1, RK2 mob + , tra +
KmR ApR; oriR6K
KmR ApR; oriR6K, GFP
As discussed before, the selection conditions for the mutagenesis experiment just mentioned were such that they ruled out inactivation of essential and metabolic genes necessary for growth in minimal medium. Also, GFP fusions may conceal the original localization of the inserted protein (as just seen with FliC). However, random generation of fluorescent fusions of the sort discussed above pinpoints proteins that are highly expressed under physiologically relevant conditions. We argue that this may become a phenomenal tool to tackle the still standing question of gene expression sites vs. chromosomal localization [50, 51], an important issue that is beyond the scope of this paper.
We have created a synthetic plasmid composed of multiple formatted and optimized functional parts that behave as predicted -both individually and as an integrated system. To the best of our knowledge this is the first report since the early 90s that describes a fully edited genetic tool optimized and streamlined for its final applications -rather than relying on cutting and pasting naturally occurring sequences . In a nutshell, non-functional DNA sequences were trimmed-off, common restriction sites present outside the multiple cloning site inside the mobile element were eliminated and the plasmid was designed following a modular pattern in which each business sequence was flanked by non-frequent restriction sites. In this respect, the key features of pBAM1 include not only the removal of many bottlenecks that flawed utilization of many of its predecessors, but also the incorporation of a fixed standard for physical assembly and exchange, where required, of new DNA pieces while maintaining its overall layout. The modularity of the design and the origin of the parts in mobile elements, which are endowed with considerable orthogonality, enable pBAM1 for two specific applications. The first, straightforward application is the use of pBAM1 as a high-throughput mutational analysis tool . Second, more important, the new vector allows exploitation of the cargo site (Figure 1 and 2) for placing a whole collection of extra genetic gadgets for expression of heterologous genes, reporter systems and environmental markers at user's will. This includes the possibility of cloning large DNA fragments inside the mobile element for a final implantation of new traits into the chromosome of the target strain. Given the randomness and the high frequencies of such insertions, one can then select the insertion out of a large collection, which adjusts expression of the desired feature to the right level under the required operation conditions [53, 54]. Furthermore, the ease of replacement of the antibiotic resistance marker (or any other functional part) allows the same transposition/delivery system to be reused for subsequent insertions. In sum, this work shows the value of DNA synthesis and standardization of functional modules for combining in a single genetic tool many valuable properties that are otherwise scattered in various vectors and rendered useless for the lack of fixed assembly formats. We anticipate pBAM1 to become one frame of reference for the construction of a large number of vectors aimed at deployment of heavily engineered genetic and metabolic circuits.
Strains, plasmids and media
The bacterial strains and plasmids used in this study are listed in Table 3. Bacteria were grown routinely in LB (10 g l-1 of tryptone, 5 g l-1 of yeast extract and 5 g l-1 of NaCl). E. coli cells were grown at 37°C while P. putida was cultured at 30°C. Selection of P. putida cells was made onto M9 minimal medium plates  with citrate (2 g l-1) as the sole carbon source. Antibiotics, when needed, were added at the following final concentration: ampicillin (Ap) 150 μg ml-1 for E. coli and 500 μg ml-1 for P. putida, kanamycin (Km) 50 μg ml-1 and chloramphenicol (Cm) 30 μg ml-1 for both species. 5-bromo-4-chloro-3-indolyl- β-D-galactopyranoside (Xgal) was added when required at 40 μg ml-1. The Pu-lacZ fusion of P. putida MAD1 (Table 3) was induced by exposing cells to saturating m-xylene vapors.
Standard procedures were employed for manipulation of DNA . Plasmid DNA was prepared using Wizard Plus SV Minipreps (Promega) and PCR-amplified DNA purified with NucleoSpin Extract II (MN). Oligonucleotides were purchased from SIGMA. For colony PCR a fresh single colony was picked from a plate and transferred directly into the PCR reaction tube. Transposon insertions were localized by arbitrary PCR of genomic DNA . Single colonies were used as the source of the DNA template for the first PCR round, which was programmed as follows: 5 minutes at 95°C, 6 cycles of 30 s at 95°C, 30 sec at 30°C, and 1 min and 30 s at 72°C; 30 cycles of 30 s at 95°C, 30 s at 30°C and 1 min and 30 s at 72°C. This was followed by an extra extension period of 4 min at 72°C. The primers used for the first round included ARB6 in combination with either ME-O-extF or ME-I-extR/GFP-extR (described in Table 2). 1 μl of the resulting product was then used as template for the second PCR round, using with the following conditions: 1 min at 95°C, 30 cycles of 30 s at 95°C, 30 sec at 52°C and 1 min and 30 sec at 72°C, followed by an extra extension period of 4 min at 72°C. The second round was performed with ARB2 and ME-O-intF or ME-I-intR/GFP-intR (Table 2). PCR reaction mixtures were purified and sequenced with either ME-O-intF or ME-I-intR/GFP-intR primers. DNA sequences were visually inspected for errors and analyzed using the Pseudomonas Genome Databasev2 (http://www.pseudomonas.com) and blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to map the precise transposon insertion point. To ascertain the conservation level of the 9-bp target sequence of Tn5 transposase we made use of WebLogo 3, a web based application available at http://weblogo.berkeley.edu/logo.cgi. DNA synthesis was outsourced from Geneart (http://www.geneart.com). The nucleotide sequences of the pBAM1 and pBAM1-GFP plasmids were submitted to the GenBank database (http://0-www.ncbi.nlm.nih.gov.brum.beds.ac.uk/genbank/) under the corresponding accession numbers HQ908071 and HQ908072.
Suicide delivery of mini-transposons
pBAM1 and its derivatives were entered into target cells by either mating or electroporation. In the first case, the plasmid was mobilized from E. coli CC118λpir (pBAM1) donor cells into Pseudomonas putida (KT2440 or MAD1 strains, Table 3) with the assistance of the helper strain E. coli HB101 (pRK600). To this end, cells were grown overnight with the appropriate antibiotics. Cells were washed with 1.0 ml of 10 mM MgSO4 and mixed in 1:1:1 ratio into 5 ml of 10 mM MgSO4 solution to obtain a final OD600 of 0.03 (3 × 107 cells) of each strain. Then, the tri-parental mating mixture was concentrated and laid onto a Millipore filter disk (0.45 μm pore-size, 13-mm diameter). The filters were incubated at 30°C onto the surface of LB agar plates. At the desired incubation time, the filter was transferred to a 5 ml of a 10 mM MgSO4 solution and vortexed to re-suspend the cells. Afterwards, appropriate dilutions were plated onto adequate selective medium as indicated for counter-selecting the donor cells in the mating. Alternatively, P. putida electrocompetent cells were prepared following the protocol described in . In this case, 100 ng - 500 ng of pBAM1 plasmid DNA were added to a 100 μl aliquot suspension containing a total of 6 × 1010 cells. The mixture was then transferred into a 2 mm gap width cuvette and electroporated with the settings of a single pulse of 2.5 kV (field strength of 12.5 kV cm-1) with a time constant of ~5 msec using program EC2 in a MicroPulser™ (BioRad). Following electropulsing, cells were quickly supplemented with 1 ml of LB and incubated at 30°C for 1 h. Then, adequate dilutions of such a suspension were plated onto M9-citrate medium plus Km for selection of mini-transposon insertions. Whether from conjugation or from electroporation, KmR clones were streaked out, single colonies checked for the loss of the plasmid marker (ApR), and the genomic DNA adjacent to the sites of insertion sequenced as explained above.
Fluorescence detection methods
Bacterial colonies growing on agar plates were inspected for emission of green fluorescence born by GFP by illumination with a 470 nm light (Safe Imager™ blue light transilluminator, Invitrogen). For visualization of GFP in individual bacteria, P. putida cells were grown up to stationary phase either in minimal M9-citrate medium or in LB. 12 ml of the cultures diluted to an OD600 of 0.5 were applied to a poly-L-Lysine-padded microscope slide and covered with mounting media for fluorescence Vectashield (Vector laboratories Inc.). Preparations were imaged with an Olympus BX61 microscope. Pictures were taken with a 100x immersion oil lens and an Olympus U-MNIBA2 filter (excitation filter 470/20 nm, emission filter 515/35 nm, beam splitter 505LP) to record fluorescence signals.
We thank members of the de Lorenzo Lab for helpful criticisms to this manuscript, Juan Carlos Martínez for technical assistance and Angel Cebolla for support and discussions. This work was defrayed by generous grants of the CONSOLIDER program of the Spanish Ministry of Science and Innovation, by the BACSIN and MICROME Contracts of the EU and by funds of the Autonomous Community of Madrid.
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