Design and validation of a transposon that promotes expression of genes in episomal DNA

Journal Pre-proof Design and validation of a transposon that promotes expression of genes in episomal DNA Alvaro Mongu´ı, Gabriel L. Lozano, Jo Handelsman, Silvia Restrepo, Howard Junca

PII:

S0168-1656(20)30008-0

DOI:

https://doi.org/10.1016/j.jbiotec.2020.01.007

Reference:

BIOTEC 8584

To appear in:

Journal of Biotechnology

Received Date:

14 September 2019

Accepted Date:

15 January 2020

Please cite this article as: Mongu´ı A, Lozano GL, Handelsman J, Restrepo S, Junca H, Design and validation of a transposon that promotes expression of genes in episomal DNA, Journal of Biotechnology (2020), doi: https://doi.org/10.1016/j.jbiotec.2020.01.007

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.

Design and validation of a transposon that promotes expression of genes in episomal DNA

Alvaro Monguí1,2*, Gabriel L. Lozano3,4,5, Jo Handelsman3,4, Silvia Restrepo6, and Howard Junca7

1

Molecular Biotechnology, Corporación CorpoGen, Bogotá, Colombia

2

Department of Biological Sciences, Universidad de los Andes, Bogotá, Colombia

3

Wisconsin Institute for Discovery and Department of Plant Pathology, University of Wisconsin,

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Madison, WI, USA Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT,

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USA

Current affiliation, Divisions of Infectious Diseases and Gastroenterology, Boston Children's

Hospital, Boston, MA, USA

Laboratory of Mycology and Plant Diseases, Universidad de los Andes, Bogotá, Colombia

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RG Microbial Ecology: Metabolism, Genomics & Evolution, Microbiomas Foundation, Chía,

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6

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Colombia

*

address: [email protected]

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Highlights

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Corresponding author: Universidad de los Andes, Carrera 1 No. 18A-12, Bogotá, Colombia. Email

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TnC_T7 transposon was constructed to improve the efficiency of metagenomic functional screenings.

TnC_T7 promotes inducible transcription of foreign DNA by expressing the T7RNA

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

polymerase.

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Random T7 promoter insertion by TnC_T7 was validated in both plasmid and fosmid DNA.

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TnC_T7 favored gene expression by inserting up to 8.7 kb upstream from the gene of interest.

Abstract Functional metagenomics, or the cloning and expression of DNA isolated directly from environmental samples, represents a source of novel compounds with biotechnological potential. However, attempts to identify such compounds in metagenomic libraries are generally inefficient in part due to lack of expression of heterologous DNA. In this research, the TnC_T7 transposon was developed to supply transcriptional machinery during functional analysis of metagenomic libraries.

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TnC_T7 contains bidirectional T7 promoters, the gene encoding the T7 RNA polymerase (T7RNAP),

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and a kanamycin resistance gene. The T7 RNA polymerase gene is regulated by the inducible arabinose promoter (PBAD), thereby facilitating inducible expression of genes adjacent to the

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randomly integrating transposon. The high processivity of T7RNAP should make this tool particularly useful for obtaining gene expression in long inserts. TnC_T7 functionality was validated by

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conducting in vitro transposition of pKR-C12 or fosmid pF076_GFPmut3*, carrying metagenomic

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DNA from soil. We identified transposon insertions that enhanced GFP expression in both vectors, including insertions in which the promoter delivered by the transposon was located as far as 8.7 kb from the GFP gene, indicating the power of the high processivity of the T7 polymerase. The results

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gathered in this research demonstrate the potential of TnC_T7 to enhance gene expression in functional metagenomic studies.

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Keywords: Functional metagenomics, Transposon mutagenesis, GFP expression, T7 promoter

1.

Introduction It is estimated that about 1% of all microorganisms present in natural environments are

cultivable under standard laboratory conditions. Consequently, the unculturable microbial world holds great potential for discovery of novel proteins and small molecules for industrial uses (Rappe and Giovannoni, 2003). Metagenomics provides an alternative to conventional microbiological

analysis. This strategy is based on DNA extraction directly from an environmental sample (metagenome) and its subsequent cloning into readily culturable bacteria (Handelsman et al., 1998). Sequence analysis of metagenomic libraries indicates that many contain genetic information for production of novel enzymes and metabolites, but functional analyses are often unsuccessful because of the limitations of heterologous gene expression of DNA from anonymous organisms in domesticated bacteria (Uchiyama and Miyazaki, 2009).

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The efficiency of heterologous gene expression in the selected host depends on the match

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between host machinery and the signals in the target DNA (Ekkers et al., 2012). Efforts to overcome the barriers have focused on varying the host species, cloning vector, origins of replication in shuttle

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vectors, genetic modifications in hosts to increase foreign ribosome binding site recognition, and chaperone co-expression to facilitate protein re-folding (Ekkers et al., 2012; Uchiyama and Miyazaki,

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2009). The rate of discovery remains low, likely due to low levels of gene expression in metagenomic

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libraries (Beloqui et al., 2008; Craig et al., 2010). A modeling approach predicted that about 40% of the enzymatic activities encoded by 32 prokaryotic genomes should be expressed in E. coli (Gabor et al., 2004). This means that a significant fraction of genes from these genomes (27-93%) would be

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invisible for the transcriptional and translational machineries of the bacterial host, and the proportion would be substantially higher among genes from environmental microorganisms that are more distantly related to E. coli. More recently it was shown that E. coli could globally transcribe

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only half of genes from Haemophilus influenzae (a closely related member of the gammaProteobacteria), a smaller proportion of genes from Pseudomonas aeruginosa (more distantly related), and fewer genes from the human genome (Warren et al., 2008). The highly expressed genes in each case had primarily promoter regions with high similarity to the sigma-70 subunit (RpoD) recognition sites of the bacterial RNA polymerase. These results indicate that transcription is the critical barrier to achieving heterologous gene expression from metagenomic libraries.

Transposons have proven useful for diverse purposes including mutagenesis, genomic manipulation, transgenesis, gene therapy, and functional modulation of gene expression in genomic and metagenomic contexts (Ivics et al., 2009; Kim et al., 2016; Leggewie et al., 2006; Reznikoff and Winterberg, 2008). In this study, we developed and validated a transposon-based tool, Mu-derived transposon TnC_T7, to increase gene expression in metagenomic libraries. This mobile element promotes heterologous gene transcription using random delivery of bi-directional T7 promoters and

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the gene encoding T7 RNA polymerase under an inducible promoter in a plasmid and a fosmid

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carrying metagenomic DNA. TnC_T7 will be a useful tool to increase gene expression in

Material and methods

2.1. Bacterial strains and growth conditions

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metagenomic libraries.

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The bacterial strains used in this study (Table 1) were grown in Luria-Bertani (LB) medium at 37°C, with constant agitation. Depending on the episomal DNA, the following antibiotics were used at the concentrations indicated: chloramphenicol 20 μg/mL, ampicillin 100 μg/mL, gentamicin 40

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μg/mL and kanamycin 40 μg/mL. Miniprep extractions were performed after overnight incubation of the bacterial cultures with the appropriate antibiotic selection.

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2.2. TnC_T7 transposon design

One end of the transposon, which included the R1 and R2 MuA transposase binding sites

and the T7 promoter region, was chemically synthesized by an external provider (Genscript, Piscataway, USA) and subsequently cloned in the unique EcoRI and BamHI restriction sites of pUC57 (pUC57_Tn). The kanamycin resistance gene with its promoter was amplified by PCR in two DNA fragments from pKD4 plasmid (Datsenko and Wanner, 2000) in order to replace the BglII restriction

site for SpeI. The first fragment was obtained using the primer sequences

5’-

CGGGATCCTTTTATGGACAGCAAGCGAACC-3’ and 5’-GACTAGTTGATCCCCTGCGCCATC-3’, while the second fragment was amplified using the primers 5’-GACTAGTGATCAAGAGACAGGATGAGG-3’ and 5’-GGCGCGCCATATCCTCCTTAGTTCCTATTCCG-3’. After ligation of the previously digested DNA fragments, the final product was inserted into AscI and BamHI restriction sites of pUC57_Tn, generating the pUC57_Tn_kanAB vector. The T7RNA polymerase (T7RNAP) encoding gene was

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amplified from BL21 (DE3) E. coli strain (Invitrogen, Carlsbad, USA) using the oligos 5’-

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ATGCCATAGCATTTTTATCC-3’ and 5’- GATTTAATCTGTATCAGG-3’, and cloned into the SacI site of plasmid pBAD18-Cm (Guzman et al., 1995) (pBAD18-Cm_t7rnap). T7RNAP coding gene plus the

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inducible-arabinose promoter (PBAD) of the vector were amplified by PCR using the oligos 5’GGCGCGCCCATTAAACGAGTATCCCG-3’ and 5’- GGCGCGCCCATAGAATTCTCGTATTGATT-3’, and the

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product was inserted into the unique AscI restriction site of pUC57_Tn_kanAB, giving rise to

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pUC57_Tn_kanAB_t7 plasmid. The other end of the transposon, which also included another R1 and R2 MuA transposase binding site and the second T7 promoter region was obtained by PCR from the vector pUC57_Tn and the amplicon further inserted into the BamHI restriction site of

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pUC57_Tn_kanAB_t7. Each intermediate step to obtain this final construct (pUC57_TnC_T7) was confirmed by colony PCR, restriction enzyme digestion, and sequencing.

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2.3. In vitro transposition

The TnC_T7 transposon was gel purified from pUC57_TnC_T7 after being digested with BglII

enzyme. Transposition reactions in vitro were performed with the MuA transposase enzyme (Thermo Fisher Scientific, Waltham, Mass, USA). Briefly, 60 ng of TnC_T7 was mixed with 200 ng of plasmid or 500 ng of fosmid DNA, 0.11 µg of MuA transposase and transposition buffer 1X. All the

reactions were brought to 10 µL with H2O and incubated for 3 hours at 30 °C, followed by heatinactivation for 10 min at 75 °C. pKR-C12 plasmid (Riedel et al., 2001) was used as the initial transposition target. The reaction was transformed into E. coli Epi300 bacterial strain (Illumina Inc., San Diego, CA, USA) using gentamicin and kanamycin as selection markers. Bacterial clones post-transposition were analyzed to identify the TnC_T7 insertion sites on the pKR-C12 vector, carrying out Sanger sequencing analysis

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from primers annealing in the transposon sequence. With this information, pKR-C12 sequence (not

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reported previously) was assembled using Geneious software (Version 4.8.5; Biomatters [https://www.geneious.com]).

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To test TnC_T7 activity in metagenomic DNA, a modified version of pCC2FOS_F076 vector was used (see below). The fosmid carried a metagenomic insert derived from soil in agricultural

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plots planted with potatoes in the Colombian Andes (Proyecto CIMA, Ministerio de Agricultura,

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República de Colombia). Fosmid sequence was acquired with 454-FLX technology (Selah Genomics, Greenville, SC, USA) and assembled and kindly provided by JC García-Betancur (Molecular Biotechnology - CorpoGen, Bogotá, Colombia & IMIB - University of Würzburg, Germany). The

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GFPmut3* (Andersen et al., 1998) gene on pKR-C12 was amplified using the following forward and reverse primers: 5’-gcgcgccAGTACTTCGGCCTGAAA-3’ and 5’-ggcgcgccTAGCTCCTGAAAATCTCG-3’, respectively (AscI restriction sites shown in lower case). This amplicon, which included an upstream

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ribosome binding site (RBS), was cloned into the unique AscI restriction site of pCC2FOS_F076 to generate pF076_GFPmut3* fosmid. This DNA construct was used as a template for TnC_T7 transposition. Transposition reactions in pF076_GFPmut3* were transformed by electroporation into E. coli Epi300 strain and the resulting clones selected with chloramphenicol and kanamycin. Sanger sequencing was performed in order to identify the transposon insertion sites on both plasmid and fosmid DNA.

2.4. Fluorescence detection TnC_T7 post-transposition clones of E. coli Epi300 pKR-C12 were each grown in LB medium to 0.4 OD600nm when they were induced with 0.2% L-arabinose for 5 hours at 30°C. As negative and positive fluorescence controls the E. coli Epi300 pKR-C12 was incubated in the absence and presence of 5 μM N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) (Sigma-Aldrich, St. Louis, MO,

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USA), a metabolite that induces expression of GFP in these constructs, based on the activation of

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LasR (Riedel et al., 2001). The fluorescence detection assays by spectrophotometry were performed in a Synergy Microplate Reader (BioTek, Winooski, VT, USA). Each bacterial culture was evaluated in

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triplicate in 96-well black polystyrene plates with clear bottoms (Sigma-Aldrich, St. Louis, MO, USA) and analyzed with an excitation wavelength of 474 nm and emission at 515 nm. Fluorescence was

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expressed in terms of Relative Fluorescence Units (RFUs)/OD600nm.

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Phenotypic analyses in LB-agar plates with 0.2% arabinose were performed for the E. coli Epi300 harboring the pKR-C12 or the pF076_GFPmut3* fosmid, after incubation of the cultures at 37°C for 14-16 hours. Fluorescence was detected using the IVIS 200 in vivo Imaging System

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(PerkinElmer, Waltham, USA) with the GFP excitation and emission filters and 15 seg of luminescent exposure. Background was corrected based on the auto-fluorescence signal of the negative control

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used in each case.

3.

Results

3.1. Construction of TnC_T7 transposon TnC_T7 transposon was assembled on a high-copy vector. The construct carries the R1 and R2 MuA transposase binding sites, the T7 promoter region, T7RNAP encoding gene with an arabinose-inducible promoter (PBAD). In addition, the vector contains a second MuA transposase

recognition site and a second T7 promoter region adjacent to the kanamycin resistance cassette (Fig. 1). The transposon can be released from pUC57_TnC_T7 by BglII restriction digestion (4,575 bp), making it ready for in vitro reactions with the MuA transposase and any episomal target DNA. Digestion with BglII is essential to generate the required nucleotide 5'-overhangs for an efficient Mu transpososome core assembly and stability as well as for the strand-transfer reactions (Savilahti et al., 1995). The integrity of each of the transposase recognition sites, the correct orientation of the

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T7RNA polymerase coding gene was confirmed by sequence analysis.

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T7 promoter regions (to be read "out" of the transposon), the kanamycin resistance gene, and the

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3.2. TnC_T7 activity validation in plasmid DNA

To determine whether the TnC_T7 transposon could enhance the expression of genes in

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episomal DNA, transposition events were performed on pKR-C12 (Riedel et al., 2001). This plasmid

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carries GFPmut3* which is under control a quorum sensing system activated when the transcriptional regulator LasR interacts with a long-chain N-Acyl homoserine lactone (e.g. 3-oxoC12-HSL), which subsequently initiates transcription from PlasB promoter (Jimenez et al., 2012). E.

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coli cannot normally express GFP in this construct because it does not synthesize the homoserine lactone signal (Gray and Garey, 2001), making it the ideal background in which to test whether TnC_T7 transposition can enhance transcription initiated from its T7 promoters.

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After transposition reactions on pKR-C12 were completed and the reactions transformed in E. coli Epi300, fluorescence of 121 randomly selected clones was analyzed by spectrophotometry. Two clones expressed significantly higher fluorescence than the no-transposition-plasmid control (data not shown). Fluorescence imaging on an additional 40 bacterial clones revealed two more clones that expressed GFP. Therefore, insertion of TnC_T7 can induce gene expression in plasmid DNA (Fig. 2). Validation of this genetic tool in an E. coli strain that does not carries the gene encoding

T7RNAP in its genome, indicates that T7RNAP expression was driven, as predicted, from its corresponding gene located within the transposon.

3.3. TnC_T7 validation in metagenomic DNA We modified the fosmid pCC2FOS_F076 by inserting the GFPmut3* gene in a unique restriction site of the metagenomic insert, to test the activity of TnC_T7 in a large metagenomic

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insert (35.6 kb) clone (Fig. 3A). We found GFP-expressing clones with the promoter delivered by the

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transposon as far as 8.7 kb upstream of GFPmut3* gene. The frequency of metagenomic clones emitting fluorescence due to insertion of the transposon was much higher than with pKR-C12 (22.4

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to 36.7% of bacterial clones post-transposition per assay expressed GFP) (Fig. 3B and 3C). From the transposition reaction on pF076_GFPmut3* fosmid DNA, 17 clones were randomly chosen based on

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their high fluorescence levels compared with the negative control, as well as 18 clones that did not

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emit fluorescence, to determine the exact site of insertion by sequencing. TnC_T7 insertions that promoted GFP expression were located between 710 bp and 8,777 bp upstream from the start

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codon of the GFPmut3* gene.

Discussion

The potential of functional metagenomics has not been realized due to the low frequency

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of expression of genes from the environmental in cultured organisms. Evidence suggests that the major block in gene expression is transcription, so we designed a universal strategy to overcome this barrier. Here we present the construction and validation of a transposon that delivers a T7 promoter and the gene encoding its cognate RNA polymerase to metagenomic DNA. We tested the system with metagenomic DNA carrying the gene encoding GFP and sought insertions that enabled expression of fluorescence.

Among the alternatives to enhance transcription of foreign genes in metagenomic libraries is use of inducible and bidirectional promoters on cloning vectors (plasmids, fosmids or cosmids) (Lämmle et al., 2007; Lussier et al., 2011). However, the long DNA fragments (>30 kb) typical in metagenomic libraries render these promoters minimally useful as they only transcribe genes proximal to the promoter. To accommodate this drawback some metagenomic expression libraries have been built with small DNA inserts (<10 kb), which enhances the chance that a cloned gene will

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be expressed but reduces the amount of DNA cloned or the chance of capturing complete pathways.

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We found clones expressing GFP that contained the insertion with the T7 promoter as far as 8.7 kb upstream of the GFP gene. This work validates the application of a tool to enhance gene expression

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in metagenomic DNA, and the continued use of long insert metagenomic libraries, such as fosmids. Transposons such as the MuExpress system are important tools to increase the expression

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of genes in metagenomic libraries (Leggewie et al., 2006; Troeschel et al., 2010). Our work aimed to

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increase the host range of a MuExpresss by integrating the T7RNAP inside the transposon, allowing its use in any E. coli background, not only in BL21 in which the T7RNAP gene is integrated in the genome. In addition, we identified and corrected a mistake made in the original MuExpress in which

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only one of the T7 promoters is active because the other was oriented in 3’-5’ sense. Indeed, we observed the GFP expression of clones from both T7 promoters. Our research was aimed at constructing and validating a novel Mu-type transposon, the

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TnC_T7 with improved properties. The results gathered here demonstrated that the TnC_T7 transposon efficiently enhanced the functional expression of genes on both plasmid and fosmid DNAs. This tool contains three key advances. First, the two T7 promoters located within the transposon were fully operative, thereby doubling the chance of obtaining gene expression compared with a uni-directional promoter. Second, transcription was initiated by the T7RNAP encoded within transposon, meaning that the E. coli host need not be a strain such as BL21, which

carries a copy of the T7RNAP gene. This is particularly important for metagenomic libraries constructed in pCC1FOS and pCC2FOS because they must be maintained in E. coli strain Epi300 to take advantage of their copy number control feature, which can be useful for preparing DNA for sequencing or enhancing accumulation of gene products. Third, expression of the gene encoding T7RNAP can be finely controlled with different D-glucose or L-arabinose concentrations for its repression or induction, respectively. This regulatory system based on PBAD is useful in situations

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that require a tight repression (such as when genes express products that are toxic to the host) or

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strong expression (Narayanan et al., 2011), both important considerations in metagenomic research.

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Although pF076_GFPmut3* is about 5 times larger than pKR-C12, the long metagenomic segment in the fosmid DNA harboring the transposon insertions that enabled GFP expression

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explained the high frequency of fluorescent clones detected. The high processivity of T7RNA

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polymerase, which can extend up to 15 kb-long transcripts (Mookhtiar et al., 1991; Muller et al., 1988), provides an outstanding size of DNA target for transcription but also means that the gene of interest may be quite distant from the transposon insertion, so direct sequencing from the insertion

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may not be a very efficient approach to identify the active gene (Leggewie et al., 2006); this may be accomplished more efficiently by sub-cloning or transposon mutagenesis. The results gathered in this research suggest that to assess the improvement of functional

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expression in metagenomic clones using TnC_T7 transposon it will be needed to screen at least 3 to 5 bacterial clones post-transposition, for each bacterial clone from the original metagenomic library. But is also important to recognize that GFP-fluorescence detection by the Imaging System used in this study are of high sensitivity. This means that detection of phenotypes by direct methods commonly used in laboratory (e.g. colony pigmentation, irregular colony morphology or halo formation on plate overlays) could involve analyzing a larger number of clones post-transposition

to recover the desired functions. This factor is particularly important since direct detection methods exhibit low resolution or sensitivity, but joint information from additional detection strategies on metagenomic clones may provide additional clues about the potential activities derived from the environmental DNA (Ekkers et al., 2012).

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Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments

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The authors want to thank the Departamento Administrativo de Ciencias, Tecnología e Innovación (Colciencias), República de Colombia; Convocatoria 489 – 2009, Código 657048925406, Contrato de

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financiación RC. 427 – 2009 Colciencias – CorpoGen, “Platform for exploring antimalarial

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compounds in metagenomes” for financial support. HJ thanks Fernando Díaz, Yakelinda Rojas, Luz Marina Olarte, Walter Ocampo and Luz Karime Afanador for technical and administrative staff

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support. AM acknowledge financial support through “Programa de Asistencias Graduadas” by Universidad de los Andes, Bogotá, Colombia.

References

Jo

Andersen, J.B., Sternberg, C., Poulsen, L.K., Bjorn, S.P., Givskov, M., Molin, S., 1998. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240–6.

Beloqui, A., de Maria, P.D., Golyshin, P.N., Ferrer, M., 2008. Recent trends in industrial microbiology. Curr

Opin

Microbiol

[pii]10.1016/j.mib.2008.04.005

11,

240–248.

https://doi.org/S1369-5274(08)00055-6

Craig, J.W., Chang, F.-Y., Kim, J.H., Obiajulu, S.C., Brady, S.F., 2010. Expanding small-molecule functional metagenomics through parallel screening of broad-host-range cosmid environmental DNA libraries in diverse proteobacteria. Appl. Environ. Microbiol. 76, 1633–41. https://doi.org/10.1128/AEM.02169-09 Datsenko, K.A., Wanner, B.L., 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12

using

PCR

products.

Proc.

Natl.

Acad.

Sci.

U.

A.

97,

6640–5.

of

https://doi.org/10.1073/pnas.120163297

S.

ro

Ekkers, D.M., Cretoiu, M.S., Kielak, A.M., Elsas, J.D. van, 2012. The great screen anomaly--a new frontier in product discovery through functional metagenomics. Appl. Microbiol. Biotechnol.

-p

93, 1005–20. https://doi.org/10.1007/s00253-011-3804-3

Gabor, E.M., Alkema, W.B., Janssen, D.B., 2004. Quantifying the accessibility of the metagenome by expression

cloning

techniques.

Env.

re

random

Microbiol

6,

879–886.

lP

https://doi.org/10.1111/j.1462-2920.2004.00640.xEMI640 [pii] Gray, K.M., Garey, J.R., 2001. The evolution of bacterial LuxI and LuxR quorum sensing regulators. Microbiology 147, 2379–87.

ur na

Guzman, L.M., Belin, D., Carson, M.J., Beckwith, J., 1995. Tight regulation, modulation, and highlevel expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121– 30.

Jo

Handelsman, J., Rondon, M.R., Brady, S.F., Clardy, J., Goodman, R.M., 1998. Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem Biol 5, R245-9. https://doi.org/S1074-5521(98)90108-9 [pii]

Ivics, Z., Li, M.A., Mates, L., Boeke, J.D., Nagy, A., Bradley, A., Izsvak, Z., 2009. Transposon-mediated genome manipulation in vertebrates. Nat Methods 6, 415–422. https://doi.org/nmeth.1332 [pii]10.1038/nmeth.1332

Jimenez, P.N., Koch, G., Thompson, J.A., Xavier, K.B., Cool, R.H., Quax, W.J., 2012. The multiple signaling systems regulating virulence in Pseudomonas aeruginosa. Microbiol. Mol. Biol. Rev. 76, 46–65. https://doi.org/10.1128/MMBR.05007-11 Kim, Y.J., Kim, H., Kim, S.H., Rha, E., Choi, S.-L., Yeom, S.-J., Kim, H.-S., Lee, S.-G., 2016. Improved metagenome screening efficiency by random insertion of T7 promoters. J. Biotechnol. 230, 47– 53. https://doi.org/10.1016/j.jbiotec.2016.05.018

of

Lämmle, K., Zipper, H., Breuer, M., Hauer, B., Buta, C., Brunner, H., Rupp, S., 2007. Identification of

ro

novel enzymes with different hydrolytic activities by metagenome expression cloning. J. Biotechnol. 127, 575–92. https://doi.org/10.1016/j.jbiotec.2006.07.036

functional

expression

of

DNA

-p

Leggewie, C., Henning, H., Schmeisser, C., Streit, W.R., Jaeger, K.-E., 2006. A novel transposon for libraries.

Biotechnol.

123,

281–7.

re

https://doi.org/10.1016/j.jbiotec.2005.12.010

J.

lP

Lussier, F.-X., Chambenoit, O., Côté, A., Hupé, J.-F., Denis, F., Juteau, P., Beaudet, R., Shareck, F., 2011. Construction and functional screening of a metagenomic library using a T7 RNA polymerase-based expression cosmid vector. J. Ind. Microbiol. Biotechnol. 38, 1321–8.

ur na

https://doi.org/10.1007/s10295-010-0915-2

Mookhtiar, K.A., Peluso, P.S., Muller, D.K., Dunn, J.J., Coleman, J.E., 1991. Processivity of T7 RNA polymerase requires the C-terminal Phe882-Ala883-COO- or "foot" Biochemistry

Jo

30, 6305–6313. https://doi.org/10.1021/bi00239a032 Muller, D.K., Martin, C.T., Coleman, J.E., 1988. Processivity of proteolytically modified forms of T7 RNA polymerase. Biochemistry 27, 5763–71.

Narayanan, A., Ridilla, M., Yernool, D.A., 2011. Restrained expression, a method to overproduce toxic membrane proteins by exploiting operator-repressor interactions. Protein Sci. 20, 51–61. https://doi.org/10.1002/pro.535

Rappe, M.S., Giovannoni, S.J., 2003. The uncultured microbial majority. Annu Rev Microbiol 57, 369– 394. https://doi.org/10.1146/annurev.micro.57.030502.090759 Reznikoff, W.S., Winterberg, K.M., 2008. Transposon-based strategies for the identification of essential bacterial genes. Methods Mol. Biol. 416, 13–26. https://doi.org/10.1007/978-159745-321-9_2 Riedel, K., Hentzer, M., Geisenberger, O., Huber, B., Steidle, A., Wu, H., Høiby, N., Givskov, M., Molin, Eberl,

L.,

2001.

N-acylhomoserine-lactone-mediated

communication

between

of

S.,

ro

Pseudomonas aeruginosa and Burkholderia cepacia in mixed biofilms. Microbiology 147, 3249–62.

-p

Savilahti, H., Rice, P.A., Mizuuchi, K., 1995. The phage Mu transpososome core: DNA requirements for assembly and function. EMBO J. 14, 4893–903.

re

Troeschel, S.C., Drepper, T., Leggewie, C., Streit, W.R., Jaeger, K.-E., 2010. Novel tools for the

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functional expression of metagenomic DNA. Methods Mol. Biol. 668, 117–39. https://doi.org/10.1007/978-1-60761-823-2_8

Uchiyama, T., Miyazaki, K., 2009. Functional metagenomics for enzyme discovery: challenges to

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efficient screening. Curr Opin Biotechnol 20, 616–622. https://doi.org/S0958-1669(09)001244 [pii]10.1016/j.copbio.2009.09.010 Warren, R.L., Freeman, J.D., Levesque, R.C., Smailus, D.E., Flibotte, S., Holt, R.A., 2008. Transcription

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of foreign DNA in Escherichia coli. Genome Res 18, 1798–1805. https://doi.org/gr.080358.108 [pii]10.1101/gr.080358.108

Fig. 1. Scale outline of TnC_T7 transposon into pUC57 vector. Light gray shows the two MuA transposase binding sites adjacent to a T7 promoter. Italics show the restriction sites involved in the construction of the transposon. The distance between the two BglII restriction sites is 4,575 bp. PBAD,

arabinose-inducible promoter; T7RNAp, T7RNA polymerase coding sequence; Kan, Kanamycin

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resistance gene.

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Fig. 2. TnC_T7 transposon insertions in pKR-C12 and fluorescence detection. (A) scale diagram of pKR-C12 plasmid. The plasmid fragment highlighted shows the exact location of the transposon

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insertions located nearby. Segmented arrows oriented from top to bottom represent transposon

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insertion sites in which the t7rnap gene is located in the sense strand of the target DNA, while the arrows from bottom to top represent the same gene located in the antisense DNA strand. pBBR1,

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replication origin of the plasmid; LasR, transcriptional regulator; P-O, Lac promoter and Lac operator system; GFP, green fluorescent protein; CmR, chloramphenicol resistance gene; GmR, gentamicin

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resistance gene; CAP, catabolite activator protein binding site; *, two independent TnC_T7 insertions in the same position. (B) fluorescence detection by spectrophotometry in terms of RFUs (GFP)/Optical density (OD); 1-8, bacterial clones post-transposition showed in A; (-), Negative control of GFP expression; (+), Positive control of GFP expression. (C) Fluorescence by the IVIS

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Imaging System detector; 1-8, bacterial clones post-transposition showed in A.

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Fig. 3. Fluorescence detection in bacterial clones with pF076_GFPmut3* after transposition. (A)

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fluorescence detection using the Imaging System for E. coli Epi300 clones transformed with the

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transposition reaction of TnC_T7 in pF076_GFPmut3*. Patches of negative and positive controls of GFP expression are shown, corresponding to clones 2 and 4 of E. coli Epi300 pKR-C12 post-

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transposition (Fig. 2C), respectively. (B) validation of GFP expression for bacterial clones with pF076_GFPmut3* fosmid using the Imaging System after their recovery in LB-agar with 20 μg/mL

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chloramphenicol, 40 μg/mL kanamycin and 0.2% L-arabinose. (C) pF076_GFPmut3* scale diagram with the identified TnC_T7 transposon insertion sites. The metagenomic insert DNA sequence is highlighted, as well as the ORFs with length greater than 150 codons located on the fosmid sense strand. White arrows and boxes represent the fosmid backbone. Shaded region includes the

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transposon insertions that promoted GFP expression.

Table 1 Bacterial strains and plasmids Bacterial strain or Relevant characteristics

Source

plasmid E. coli TOP10

F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 Δ lacX74 Invitrogen recA1 araD139 Δ(araleu)7697 galU galK rpsL (StrR) endA1

E. coli Epi300

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nupG. Strain used for regular transformations. F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80dlacZΔM15 Δ lacX74 Illumina

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recA1 endA1 araD139 Δ(araleu)7697 galU galK λ- rpsL (StrR) nupG trfA dhfr. Strain used for functional expression.

F- ompT gal dcm lon hsdSB(rB- mB-) λDE3 (lacI lacUV5-T7 Invitrogen

-p

E. coli BL21 (DE3)

t7rnap.

re

gene 1 ind1 sam7 nin5). Strain used for amplification of

ApR; lacZ, cloning vector.

pUC57_Tn

ApR; pUC57 carrying one R1-R2 MuA transposase binding This work

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pUC57

Genscript

pKD4

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site and a T7 promoter.

ApR, KmR; contains an FRT-flanked kanamycin resistance (Datsenko

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gene.

pUC57_Tn_kanAB

ApR, KmR; pUC57_Tn carrying the kanamycin resistance gene from pKD4.

and Wanner, 2000) This work

pBAD18-Cm

CmR; arabinose inducible (PBAD) expression vector.

(Guzman et

al.,

1995) pBAD18-Cm_t7rnap

CmR; pBAD18-Cm carrying the T7RNAP coding sequence.

This work

pUC57_Tn_kanAB_t7 ApR, KmR; pUC57_Tn_kanAB carrying the PBAD promoter This work and the t7rnap from pBAD18-Cm_t7rnap. ApR, KmR; pUC57_Tn_kanAB_t7 carrying another R1-R2 This work

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pUC57_TnC_T7

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MuA transposase binding site and a second T7 promoter.

This construction harbors the complete TnC_T7 sequence. GmR, CmR; pBBR1MCS-5 carrying PlasB-gfp (ASV) Plac-lasR.

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pKR-C12

CmR; fosmid cloning vector.

pCC2FOS_F076

CmR; pCC2FOS with a 35.6 kb insert of metagenomic DNA

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re

pCC2FOS

(Riedel et al., 2001) Illumina 1

from agricultural soil planted with potatoes pF076_GFPmut3*

CmR; pCC2FOS_F076 carrying the RBS and the GFPmut3*

Molecular Biotechnology - CorpoGen, Bogotá, Colombia.

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1

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coding sequence from pKR-C12.

This work