Modified Tn7 transposon vectors for controlled chromosomal gene expression

ABSTRACT

Complementation remains a foundation for demonstrating molecular Koch’s postulates. While this is frequently achieved using plasmids, limitations such as increased gene copy number and the need for antibiotic supplementation to avoid plasmid loss can restrict their use. Chromosomal integration systems using the Tn7 transposon provide an alternative to plasmids for complementation and facilitate the stable insertion of genes at the chromosomal attTn7 site without the need for selection pressure. Here, we enhanced the utility of mini-Tn7 insertion vectors by the addition of inducible (Pcym) and constitutive (PcL and PrpsM) promoters, allowing differential transcriptional control of genes integrated into the chromosome. We validated the utility of these promoters by cloning the gfp gene, encoding green fluorescent protein, downstream of each promoter and integrating a mini-Tn7 construct harboring these elements into the attTn7 site on the chromosome of the Escherichia coli K-12 strain MG1655. The PcL and PrpsM promoters provided equivalent levels of GFP expression and offered flexibility based on the target host strain. Activation of the tightly regulated Pcym promoter with its inducer cumate resulted in tunable expression of GFP in a dose-dependent manner. We further demonstrated the tight control of the Pcym promoter using the toxic impCAB genes, and the expression of which is detrimental to E. coli viability. Together, these modified mini-Tn7 vectors allowing differential control of genes integrated into the chromosome at a conserved site offer an efficient system for complementation where plasmid use is restricted.

IMPORTANCE

Chromosomal integration using mini-Tn7 vectors provides an efficient means to insert genes into the chromosome of many gram-negative bacteria. Insertion occurs at a conserved site and allows for the stable integration of genes in single copy. While this system has multiple benefits for enabling complementation, a cornerstone for fulfilling molecular Koch’s postulates, greater flexibility for controlled gene expression would enhance its utility. Here, we have added to the function of mini-Tn7 vectors by the addition of inducible and constitutive promoters and demonstrated their capacity to drive the controlled expression of target genes integrated into the chromosome. In addition to complementation, these modified vectors offer broad application for other approaches including chromosomal tagging, in vivo expression, metabolic engineering, and synthetic biology.

INTRODUCTION

The molecular Koch’s postulates are well-established principles to demonstrate that a gene confers a hypothesized function, such as a role in virulence. These postulates describe that specific inactivation of a gene of interest should result in attenuation of virulence or pathogenicity, and that the reintroduction of the wild-type gene should restore virulence to wild-type levels (1). The latter of these processes, termed complementation, has been achieved primarily by cloning genes into plasmids and introducing recombinant plasmids into bacteria.

However, the use of plasmids for complementation comes with several disadvantages. Plasmids must be maintained through selective pressure, which is primarily achieved via antibiotic supplementation. Antibiotics can be challenging to administer in vivo, may not be desirable in vitro, and generate increased economic burden and environmental/waste concerns for industrial applications (2). Plasmid-based expression systems suffer from both structural and segregational instability, resulting in significant productivity and economic losses in industrial settings (3, 4). The multicopy nature of plasmids often increases gene dosage, which may confer abnormal phenotypes compared to the wild-type strain, particularly when the wild-type gene is present as a single copy on the chromosome. In contrast, chromosomally inserted genes are genetically stable and single-copy, thereby reducing metabolic burden and cell-to-cell variation (5).

The extensively studied bacterial transposon Tn7 has been exploited as an alternative complementation approach. Transposons are DNA sequences capable of moving between different parts of the genome, which is mediated by a self-encoded transposase enzyme. The Tn7 transposase comprised TnsA and TnsB subunits, which recognize binding sites within the left (Tn7L) and right (Tn7R) ends of the transposon. TnsA is responsible for cleaving the 5′ ends of Tn7, while TnsB cleaves and joins the 3′ ends to target DNA, thereby resulting in orientation-specific insertion into target DNA (6–9). The transposition regulator TnsC and DNA-binding protein TnsD coordinate and direct the transposase to a specific site on the chromosome, termed the attachment site (attTn7). TnsD recognizes a sequence in the 3′ end of the highly conserved and essential chromosomal glmS gene, which encodes glutamine synthetase involved in N-acetylglucosamine biosynthesis (10, 11). Transposition of Tn7 downstream of the glmS coding sequence (CDS) ensures that glmS expression is not disrupted. This facilitates coexistence between Tn7 and its bacterial host, with no unfavorable effects to the host cell identified in previous studies (8, 12).

The mini-Tn7 derivative and transposase expression vectors developed by Crépin et al. possess a broad host range and enable efficient Tn7 transposition into attTn7 in Escherichia coli and other Enterobacteriaceae (13). However, it is often desirable to control the level of expression of genes inserted into the chromosome. This requires the addition of a promoter, representing an additional step and increased complexity in the construction of the vector. Importantly, the validation of an existing set of different promoters at attTn7 would improve the feasibility of expressing genes from the chromosome, given that the level of gene expression can vary widely (14).

Here, we have enhanced the utility of the mini-Tn7 derivative vector with the addition of the inducible cumate promoter Pcym, as well as the constitutive PcL and PrpsM promoters, facilitating various levels of transcriptional control for genes integrated into attTn7. The utility of these constructs was validated by cloning the gfp gene encoding the green fluorescent protein downstream of these promoters and integration into the chromosomal attTn7 site of the E. coli K-12 strain MG1655. We demonstrated that both the constitutive PcL and PrpsM promoters enabled the functional expression of GFP, while the activity of the inducible Pcym promoter was both tightly controlled and titratable in driving chromosomal GFP expression. The tight regulation of Pcym was further validated by its regulation of the toxic impCAB operon, whose expression conferred a severe growth defect, while cell viability was unchanged without induction.

RESULTS

Construction of mini-Tn7 derivative vectors comprising different promoters

The mini-Tn7 derivative vector pGP-Tn7-Cm described by Crépin et al. is a highly mobilizable suicide vector harboring the gamma origin of replication from plasmid R6K and the conjugative transfer mob from RP4 (Fig. 1). Tn7 encoded within this vector harbors a chloramphenicol resistance cassette (cat gene) allowing for the selection of Tn7 in the vector following integration into the chromosome. Flippase recognition target (FRT) sites flank the chloramphenicol cassette, allowing flippase recombinase protein-mediated excision of the resistance cassette from the chromosome, a feature that alleviates biosafety and environmental concerns using this system.

FIG 1.

Schematic diagram of the mini-Tn7 derivative vector pGP-Tn7-Cm and the modified vectors comprising the inducible cumate (Pcym) promoter and the constitutive PcL and PrpsM promoters. The promoters were cloned into the MCS in Tn7, thereby generating pGP-Tn7-Pcym, pGP-Tn7-PcL, and pGP-Tn7-PrpsM. The cat gene is flanked by FRTs, which allow excision of the selection marker by FLP recombinase (not shown). Selected commonly used single-cutter restriction sites are shown. Abbreviations: bla, beta-lactamase; cat; chloramphenicol acetyl transferase (resistance); CuO, cymR operator; cymR, repressor of p-cymene catabolism; mob RP4, conjugative transfer Mob RP4; ori R6K, origin of replication; Tn7L, Tn7 left end; Tn7R, Tn7 right end.

The pGP-Tn7-Cm vector was modified by introducing a series of differentially regulated promoters to enable either tightly controlled or constitutive expression of chromosomally integrated genes. Controlled expression was mediated by the previously described cumate (p-isopropyl benzoate)-inducible promoter (hereafter termed Pcym), which combines the strong class 1 integron promoter Pc with the CymR repressor and associated cumate operators (CuO) from Pseudomonas putida (15). Transcription from the Pcym promoter is exquisitely regulated and tunable over several orders of magnitude by the addition of cumate (16, 17). In contrast, constitutive expression was driven by the well-characterized PcL promoter from bacteriophage lambda (18–20) and the PrpsM promoter from Salmonella enterica serovar Typhimurium (21).

The inducible cumate promoter Pcym comprising the Pc promoter, the cymR transcriptional repressor, and the cymR operator sequences CuO, along with the constitutive PcL promoter, were PCR amplified from existing template sequences (see Materials and Methods) and cloned into the SmaI site within the multi-cloning site (MCS) of pGP-Tn7-Cm, generating pGP-Tn7-Pcym and pGP-Tn7-PcL, respectively (Fig. 1). The constitutive PrpsM promoter was PCR amplified from an existing template sequence and cloned into the SacI and EcoRI sites within the MCS of pGP-Tn7-Cm, generating pGP-Tn7-PrpsM. The insertion of these elements harboring additional restriction sites reduced the number of available single-cutting restriction sites. To improve the utility of these plasmids for subsequent cloning steps, the original MCS was replaced with an expanded MCS harboring multiple single-cutting restriction sites. Inverse PCR was used to amplify these plasmids (excluding their existing MCS), in which the primers contained overhangs encoding single-cutting restriction sites, which were ligated together to construct an expanded MCS. The addition of Pcym did not produce as many restriction site duplications; hence, there were sufficient single-cutting restriction sites, and no further modifications to the MCS were made. The final constructs contain at least six frequently used restriction sites, thereby improving the utility of these plasmids (Fig. 1).

Cloning of gfp under the control of promoters and insertion into the chromosome

To validate the utility of our modified vectors, we cloned the gfp gene encoding the green fluorescent protein downstream of each promoter. We PCR amplified gfp from an existing template sequence (see Materials and Methods), with an optimized ribosome binding site (RBS) incorporated within the 5′-primer upstream of the GFP CDS. The amplicon was cloned downstream of each promoter, generating pGP-Tn7-Pcym-GFP, pGP-Tn7-PcL-GFP, and pGP-Tn7-PrpsM-GFP (Table 1). These vectors were transformed into E. coli GT115, which encodes the pir gene allowing the replication of suicide vectors harboring the gamma origin of replication from R6K.

TABLE 1.

Bacterial strains and plasmids

Strain or plasmid	Description	Reference
Strains
MG1655	E. coli K-12	(22)
GT115	F- mcrA Δ(mrr-hsdRMS-mcrBC) f80lacZDM15 ΔlacX74 recA1 rpsL (StrA) endA1 Ddcm uidA(DMluI)::pir-116 ΔsbcC-sbcD	InvivoGen (catalogue no. gt115-11)
β2163	E. coli strain carrying a dapA allele (which leads to diaminopimelate auxotrophy) and the integrated RP4 from SM10 in the chromosome	(23)
MS8488	E. coli-containing chromosomal PcL promoter	(18, 19)
MS24113	MG1655attTn7::Tn7-PcL-GFP	This study
MS24116	MG1655attTn7::Tn7-PrpsM-GFP	This study
MS22188	MG1655attTn7::Tn7-Pcym-GFP	This study
MS24882	MG1655attTn7::Tn7-Pcym-impCAB	This study
Plasmids
pGP-Tn7-Cm	Mini-Tn7 derivative vector	(13)
pSTNSK	Transposase expression vector harboring TnsABCD	(13)
pGP-Tn7-PcL-small_MCS	Mini-Tn7 derivative vector with cloned PcL promoter	This study
pGP-Tn7-PcL	pGP-Tn7-PcL-small_MCS with original MCS replaced with expanded MCS	This study
pGP-Tn7-PcL-GFP	pGP-Tn7-PcL with cloned gfp	This study
pGcCe	Plasmid with the S. enterica serovar Typhimurium PrpsM promoter and gfp	(24)
pGP-Tn7-PrpsM-small_MCS	Mini-Tn7 derivative vector with cloned PrpsM promoter	This study
pGP-Tn7-PrpsM	pGP-Tn7-PrpsM-small_MCS with original MCS replaced with expanded MCS	This study
pGP-Tn7-PrpsM-GFP	pGP-Tn7-PrpsM with cloned gfp	This study
pUS250	Expression vector using cumate-inducible promoter	Gift from Nicholas Coleman
pGP-Tn7-Pcym	Mini-Tn7 derivative vector with cloned Pcym promoter	This study
pGP-Tn7-Pcym-GFP	pGP-Tn7-Pcym with cloned gfp	This study
pMS7163B	IncB/O/K/Z plasmid isolated from E. coli	(25)
pGP-Tn7-Pcym-impCAB	pGP-Tn7-Pcym with cloned impCAB	This study

We then performed the chromosomal integration protocol as previously described by Crépin et al. (13) and also described in further detail in the Materials and Methods. Firstly, the mini-Tn7 vectors were extracted from GT115 and transformed into E. coli β2163, which overexpresses conjugation pili and is auxotrophic for diaminopimelic acid (DAP), therefore requiring DAP supplementation (Fig. 2). Conjugation between the donor strain β2163 harboring the mini-Tn7 plasmids and the recipient E. coli K-12 strain MG1655 harboring the Tn7 transposase expression vector pSTNSK was performed on lysogeny broth (LB) agar supplemented with DAP. Bacteria were grown overnight at 28°C to permit the replication of the temperature-sensitive pSTNSK vector. Colonies were recovered from the plate and streaked onto LB agar supplemented with chloramphenicol but not DAP, and subsequently incubated at 37°C. The absence of DAP impedes the growth of the DAP-auxotrophic β2163 donor, while incubation at 37°C facilitates the loss of the temperature-sensitive pSTNSK vector from the MG1655 recipient. Given that MG1655 does not encode the pir gene, the mini-Tn7 suicide vectors are unable to replicate in these recipient cells and are therefore cured. However, chloramphenicol supplementation in the media selects for MG1655 recipients harboring a chromosomal Tn7 insertion containing the promoter and gfp at attTn7. The resultant strains were termed MG1655attTn7::Tn7-Pcym-GFP, MG1655attTn7::Tn7-PcL-GFP and MG1655attTn7::Tn7-PrpsM-GFP. These strains contained the target gene inserted at the attTn7 site but lacked the temperature-sensitive pSTNSK vector (Fig. 2).

FIG 2.

Schematic representation of the insertion of Tn7 harboring a promoter and gfp into MG1655 attTn7. The promoter represents Pcym (cymR repressor gene not shown), PcL or PrpsM. Tn7 is orientation-specific and inserts specifically into the chromosomal attTn7 site downstream of glmS. The temperature-sensitive plasmid pSTNSK encodes the TnsABCD transposase. Abbreviations: attTn7, attachment site; attTn7 half, 5 bp half of attTn7 resulting from duplication; bla, beta-lactamase; cat; chloramphenicol acetyl transferase (resistance); Cm30, chloramphenicol 30 µg/mL; gfp, green fluorescent protein; glmS, glutamine synthetase; mob RP4, conjugative transfer Mob RP4; ori R6K, origin of replication; pstS, phosphate ABC transporter; Tn7L, Tn7 left end; Tn7R, Tn7 right end. The original pGP-Tn7-Cm vector was designed by Crépin et al. (13).

Insertion of Tn7 derivative transposons into the chromosome of colonies that grew in the presence of chloramphenicol was assessed by colony PCR using primers targeting the flanking regions of the attTn7 site. A positive insertion rate of 100% (4/4 colonies) was observed for MG1655attTn7::Tn7-Pcym-GFP and MG1655attTn7::Tn7- PcL-GFP, while a positive insertion rate of 75% (3/4 colonies) was observed for MG1655attTn7::Tn7-PrpsM-GFP (Fig. S1A and B).

Different promoters drive constitutive versus induced GFP expression

To validate the use of our modified vectors, we measured the fluorescence of the three MG1655 strains harboring each promoter and gfp at the chromosomal attTn7 site. No induction or 1 µM cumate induction of MG1655attTn7::Tn7-Pcym-GFP did not increase fluorescence compared to wild-type MG1655, demonstrating that Pcym is tightly controlled (Fig. 3A and B). In contrast, induction using 10 and 100 µM cumate significantly increased MG1655attTn7::Tn7-Pcym-GFP fluorescence, demonstrating the functionality and dosage effect of this system. The fluorescence of MG1655attTn7::Tn7-PcL-GFP and MG1655attTn7::Tn7-PrpsM-GFP was also significantly higher than wild-type MG1655, demonstrating the effectiveness of these constitutive promoters. However, their fluorescence was lower than that of MG1655attTn7::Tn7-Pcym-GFP with 10 or 100 µM induction, revealing that Pcym possesses the strongest promoter activity. There were no significant changes in optical density (OD) for any of the strains harboring Tn7 compared to wild-type MG1655 (Fig. S2A and B). Furthermore, growth was not significantly altered by different cumate concentrations. These data suggest an absence of negative effects on the bacterial host strain.

FIG 3.

Fluorescence of MG1655 strains harboring promoters and gfp at the chromosomal attTn7 site as shown by (A) kinetics and (B) area under the curve. All strains other than MG1655 have the prefix “MG1655attTn7::Tn7-” (not shown). MG1655attTn7::Tn7-Pcym-GFP was supplemented with cumate at time = 0 h. Biological replicates are shown as dots and fluorescence kinetics were performed using enhanced dynamic range. One-way ANOVA and Tukey’s multiple comparisons test were performed for area under the curve analysis.

Chromosomal insertion of Tn7 harboring promoters is genetically stable

To validate that insertion of our modified Tn7 constructs is genetically stable without the need for selective pressure, we passaged MG1655attTn7::Tn7-Pcym-GFP in LB broth 14 times over 7 days. Subsequent plating of this passaged strain on LB agar in the absence of chloramphenicol but with an addition of 100 µM cumate revealed that 100% (80/80) of randomly selected single colonies fluoresced, suggesting that Tn7 harboring the Pcym and gfp is genetically stable without antibiotic selection.

Tn7 harboring the Pcym promoter tightly regulates the expression of toxic genes

We previously demonstrated that over-expression of the impCAB operon from the IncB/O/K/Z plasmid pMS7163B adversely affects E. coli viability (25). To demonstrate that the Pcym promoter within Tn7 can tightly control genes that encode toxic products, we cloned the ~2 kb impCAB operon into pGP-Tn7-Pcym and then integrated Tn7 harboring Pcym-impCAB into the MG1655 chromosome, generating MG1655attTn7::Tn7-Pcym-impCAB. To test cell viability, overnight bacterial cultures were standardized to OD₆₀₀ = 0.5, then serially diluted 10-fold, and spotted onto LB agar with and without 75 µM cumate as an inducer. There was a severe reduction in MG1655attTn7::Tn7-Pcym-impCAB viability when grown in the presence of cumate, while wild-type MG1655 growth remained unaffected (Fig. 4). In contrast, MG1655attTn7::Tn7-Pcym-impCAB grown on LB agar (minus cumate) showed no difference in cell viability compared to wild-type MG1655, indicating that Pcym tightly controlled impCAB expression and alleviated its adverse effects (Fig. 4).

FIG 4.

Growth of MG1655 and MG1655attTn7::Tn7-Pcym-impCAB on LB agar with and without cumate supplementation. Overnight cultures were standardized to OD₆₀₀ 0.5, then serially diluted 10-fold, and spotted on LB agar or LB agar+cumate (75 µM). Photos are representative of three biological replicates.

DISCUSSION

We have modified the original mini-Tn7 derivative vector constructed by Crépin et al. with the addition of the inducible cumate promoter Pcym and the constitutive promoters PcL and PrpsM, enabling transcriptional control of genes integrated into the chromosomal attTn7 site. Our set of three modified mini-Tn7 vectors provide a simple, rapid, and validated method for the insertion of genes into the chromosome in a stable, single copy, orientation-specific, and site-specific manner that ultimately circumvents the need for antibiotic supplementation. Insertion of genes specifically into attTn7 is advantageous given the multiple strains constructed using this approach are otherwise isogenic, with no negative effects observed following the broad use of the Tn7 system. Notably, we validated expression driven by promoter activity at the attTn7 site, demonstrating that the promoters are compatible with this locus. This is important because the expression can be location-dependent (14).

Applications where antibiotic supplementation is challenging would benefit from the use of the Tn7 vectors comprising the constitutive PcL and PrpsM promoters (pGP-Tn7-PcL and pGP-Tn7-PrpsM). The Tn7 vector comprising the inducible cumate promoter Pcym (pGP-Tn7-Pcym) is well-suited for applications requiring very strong promoter activity and high expression of target genes, although its dose-dependent regulation enables its use for any desired level of gene expression. Pcym will also be beneficial for controlling the expression of inserted genes that confer negative effects on the cell (e.g., toxins), because Pcym activity is tightly controlled by its repressor protein. Indeed, we validated this application using the toxic impCAB genes.

Similar cumate-inducible promoters have been used in many different organisms, including E. coli (16), sphingomonads and other Alphaproteobacteria (26), Streptomyces and other actinobacteria (27), and even mammalian cells (15). Studies have demonstrated that the analogous cumate gene switch (16) and PQJ promoter from Pseudomonas aeruginosa (17) are tightly controlled, dose-dependent, and result in homogenous and high expression of target genes when induced with cumate. The inducible P_BAD and P_tet promoters, which are de-repressed by arabinose and anhydrotetracycline, respectively, have also been commonly used in plasmids (28, 29) and on the chromosome (30–32).

Induction using cumate offers several advantages compared to commonly used inducers. For example, the use of anhydrotetracycline is limited by its toxicity while the non-toxic isopropyl-β-d-thiogalactopyranoside (IPTG) is not economically suited for industrial or large-scale applications. Sugar inducers such as arabinose are metabolizable, potentially impacting cell physiology and resulting in the depletion of the inducer. In contrast, cumate is inexpensive, non-toxic, non-metabolizable as a carbon source, effective at low concentrations and results in dose-dependent gene regulation (33).

The original pGP-Tn7-Cm vector was optimized for the Enterobacteriaceae and was validated in nine strains across five species, encompassing E. coli, Klebsiella pneumoniae, Citrobacter rodentium, Cronobacter sakazakii, and S. enterica serovars Typhimurium and Typhi (13). Given that no modifications have been made to the origins of replication and transfer, nor to the transposition systems, the function of our plasmids in these hosts is not expected to change. Other chromosomal integration approaches, including suicide vectors, homologous recombination, and CRISPR/Cas methodologies, have also been described and possess their own advantages and limitations compared to the pGP-Tn7-Cm vector system (34). The Tn7 integration method uses two plasmids, and only one of which requires modification, presenting a straight-forward system with applicability across multiple organisms. We further demonstrated the system can effectively integrate the gfp gene (714 bp) and the impCAB gene cluster (1954 bp), which together with previous work that integrated the pstSCA gene cluster (2977 bp) (13), reveals the broad capability of the pGP-Tn7-Cm vector system for introducing DNA fragments of different sizes into the E. coli chromosome. We also showed that inserted genes were genetically stable in the absence of antibiotic selection. This is consistent with a previous work demonstrating that strains carrying different inserted genes retained their insertions in the absence of selective pressure after 14 passages (over 7 days) in LB broth and after inoculation and growth in the murine model of urinary tract infection (13).

In conclusion, our modified Tn7-promoter system combines the use of three different promoters with an established system that enables efficient integration of genes into the chromosome at a highly conserved site. We anticipate that our Tn7-promoter vectors will be useful in a variety of applications, including chromosomal complementation, chromosomal tagging, in vivo gene expression, metabolic engineering, and synthetic biology.

MATERIALS AND METHODS

Bacterial strains and growth conditions

All strains and plasmids used in this study are listed in Table 1. Plasmid pUS250 was a gift from Nicholas Coleman (Addgene plasmid # 198322; https://www.addgene.org/198322/; RRID: Addgene_198322). Bacteria were routinely cultured at 37°C or 28°C under static or shaking (250 rpm) conditions in solid or liquid LB medium, supplemented as appropriate with: chloramphenicol (30 µg/mL), gentamicin (20 µg/mL), and 2,6-DAP (Sigma-Aldrich, catalogue no. 33240) (0.3 mM). Bacterial strains were stored in 15% glycerol at −80°C.

DNA purification, analysis, and sequencing

Plasmids were purified using the QIAprep Spin Miniprep Kit (Qiagen, catalogue no. 27104). Genomic DNA was purified using the Wizard genomic DNA Purification Kit (Promega, catalogue no. A1120). DNA quality and concentration were measured using the Nanodrop 2000 Spectrophotometer (Thermo Scientific). All constructs were confirmed via Sanger sequencing, which was performed using BigDye Terminator Mix v3.1 and sequenced by the Genetic Research Services, The University of Queensland.

Construction of Tn7 transposon vectors

Primers used in this study are listed in Table S1. The PcL, PrpsM, and Pcym promoters were amplified from MS8488, pGcCe, and pUS250, respectively (Table 1), using the KAPA HiFi PCR Kit (KAPA Biosystems, catalogue no. KK2102) as per manufacturer’s instructions. The mini-Tn7 derivative vector pGP-Tn7-Cm was digested with SmaI (New England Biolabs, catalogue no. R0141), and the PcL and Pcym promoters were cloned into the SmaI site using homologous alignment cloning (HAC) in E. coli GT115 as described previously (35), generating pGP-Tn7-PcL-small-MCS and pGP-Tn7-Pcym. To clone the PrpsM promoter into pGP-Tn7-Cm, the promoter amplicon and the vector were digested with SacI (New England Biolabs, catalogue no. R3156) and EcoRI (New England Biolabs, catalogue no. R3101), followed by ligation using T4 DNA ligase (New England Biolabs, catalogue no. M0202), generating pGP-Tn7-PrpsM-small-MCS. To replace the existing MCS of pGP-Tn7-PcL-small-MCS and pGP-Tn7-PrpsM-small-MCS with a new and expanded MCS containing several single-cutting restriction sites, inverse PCR was used to amplify these plasmids (excluding their existing MCS), in which the primers contained overhangs encoding single-cutting restriction sites. The amplicons were then ligated together, generating pGP-Tn7-PcL and pGP-Tn7-PrpsM. The plasmids, pGP-Tn7-Pcym, pGP-Tn7-PcL, and pGP-Tn7-PrpsM, have been deposited into Addgene (plasmid IDs 225280-225282).

Cloning of target genes into Tn7 transposon vectors

The gfp gene and impCAB gene cluster were PCR amplified from plasmid pGcCe (24) and pMS7163B (20, 25, 36), respectively, using the KAPA HiFi PCR Kit. To clone gfp downstream of each promoter in pGP-Tn7-PcL and pGP-Tn7-PrpsM, the gfp amplicon and vectors were digested with MluI (New England Biolabs, catalogue no. R3198) and NotI (New England Biolabs, catalogue no. R3189) and then ligated together as described above. To clone gfp downstream of the Pcym promoter in pGP-Tn7-Pcym, the vector was digested with SmaI, and the gfp amplicon was cloned into the SmaI site using HAC as described previously (35). To clone impCAB downstream of the Pcym promoter in pGP-Tn7-Pcym, the impCAB amplicon and vector were digested with ApaI (New England Biolabs, catalogue no. R0114S) and XhoI (New England Biolabs, catalogue no. R0146S) and ligated as described previously.

Transformation

Electrocompetent cells were prepared, and transformations were performed as described previously (25). The plasmids pGP-Tn7-PcL-GFP, pGP-Tn7-PrpsM-GFP, pGP-Tn7-Pcym-GFP, and pGP-Tn7-Pcym-impCAB were transformed into the conjugation pili overexpressing E. coli strain β2163, generating β2163(pGP-Tn7-PcL-GFP), β2163(pGP-Tn7-PrpsM-GFP), β2163(pGP-Tn7-Pcym-GFP), and β2163(pGP-Tn7-Pcym- impCAB), hereafter referred to as the donor strains. The Tn7 transposase expression vector pSTNSK was electroporated into E. coli MG1655, generating MG1655(pSTNSK), hereafter termed the recipient strain.

Chromosomal integration of Tn7 into MG1655

The donor strains were grown overnight in 5 mL LB medium supplemented with chloramphenicol at 37°C under static conditions. An overnight culture of the recipient strain was prepared in 5 mL LB medium supplemented with gentamicin and DAP then grown at 28°C under shaking conditions. Recipient cells were then diluted 1:10 and grown for 3 h at 28°C under shaking conditions in LB medium supplemented with gentamicin and DAP. The donor and recipient cell cultures were then pelleted (3,500× g, 5 min) and gently washed with 5 mL 10 mM MgSO₄ (Sigma-Aldrich, catalogue no. M1880). Each culture was then resuspended in 500 µL 10 mM MgSO₄. Each donor was mixed with the recipient (100 µL each); then the cell mixture was pelleted (3,500× g, 5 min) and resuspended in 150 µL 10 mM MgSO₄. The mixture was spotted onto solid LB medium supplemented with DAP and incubated for 16 h at 28°C, facilitating conjugation and transposition as described previously (13). The spot was recovered in 1.5 mL phosphate buffered saline (PBS) and streaked onto solid LB medium supplemented with chloramphenicol. The bacteria were incubated at 37°C overnight to mediate the loss of pSTNSK and select for recipient cells harboring the chromosomal Tn7 insertion. To screen for positive colonies carrying the Tn7 insertion, colony PCR was performed using OneTaq DNA Polymerase (New England Biolabs, catalogue no. M0480S) as per manufacturer’s instructions.

Fluorescence and growth kinetics

Overnight cultures were grown in liquid LB medium for 16 h at 37°C under shaking conditions. The cultures were standardized to OD₆₀₀ = 0.1, and 100 µL of cells was added to 100 µL of LB medium in a Nunc MicroWell 96-Well Optical-Bottom Plate (ThermoFisher Scientific, catalogue no. 165305). Strains-harboring constitutive promoters were added to plain LB medium, while strains requiring induction were added to LB medium supplemented with 2 × 4-isopropylbenzoic acid (Sigma-Aldrich, catalogue no. 268402). Fluorescence (excitation wavelength 470 nm and emission wavelength 515 nm) and OD₆₀₀ kinetics were performed on the CLARIOstar Plus (BMG LABTECH) at 37°C with orbital shaking over 24 h and 15 min. Measurements were obtained every 15 min using the enhanced dynamic range.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aem.01556-24.

Supplemental figures. aem.01556-24-s0001.pdf.

Fig. S1, colony PCR; Fig. S2, optical density kinetics.

Table S1. aem.01556-24-s0002.xlsx.

Primer list.

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