Development of a papillation assay using constitutive promoters to find hyperactive transposases

TL;DR: An improvement of the well-known papillation assay is presented where in place of an inducible promoter, a set of constitutive promoters cloned into a one or five copies vector in presence or absence of a ribosome binding site is designed.

Abstract: Background Transposable elements is an extremely diverse group of genetic elements encoding their own mobility. This ability has been exploited as a powerful tool for molecular biology and genomics techniques. However, transposition activity is regulated by cis and/or trans mechanisms because of the need to co-exist with their host. This represents a limitation to their usage as biotechnological tools. The development of screening assays and the improvement of current ones is therefore needed to find hyperactive transposases. Results We present in this study an improvement of the well-known papillation assay where in place of an inducible promoter, we designed a set of constitutive promoters cloned into a one or five copies vector in presence or absence of a ribosome binding site. This set of vectors provides a wide range of transposase expression and offers a more uniform expression of the transposase across cells compared to inducible promoters. These constructs can therefore be used to screen for hyperactive transposases or for transposases resistant to overproduction inhibition, a mechanism affecting DNA transposases such as Hsmar1, which decreases the transposition rate when the transposase concentration increases. We characterized and validated our set of vectors with the Hsmar1 transposase and took advantage of our approach to investigate the effects on the transposition rate of inserting mutations in the Hsmar1 dimer interface or of covalently binding two Hsmar1 monomer. Conclusions This improved papillation assay should be applicable to a wide variety of DNA transposases. It also provides a straightforward approach to screen transposase mutant libraries with a specific expression level to find hypoactive, hyperactive or overproduction inhibition resistant transposases. Our approach could also be useful for synthetic biology as a combination of the wild type or covalently bound Hsmar1 transposase with a library of weak promoters offers the possibility to find promoters expressing on average one or two proteins per cell.

Summary

Summary

• 2 Background 23 Transposable elements (TEs) form a diverse group of DNA sequences encoding functions 24 for their own mobility.
• Their use is sometimes limited because their activity is 26 auto-regulated to allow them to cohabit within their hosts without causing excessive genomic 27 damage.
• They are found in virtually all organisms and are 68 particularly present in eukaryotes where they can represent a high percentage of the 69 genome (1-3).
• Thus, OPI curbs 82 Hsmar1 transposition rate to avoid damaging the host genome by excessive transposition 83 (12).
• 91 5 To facilitate the isolation of suitable transposase mutants, the papillation assay was 92 developed as an efficient screening procedure (Supplementary Figure 1) (19, 20).
• The 115 transposition rate is dependent on the concentration and activity of the transposase (12).
• The authors also compared by western blotting these constructs 169 8 with the Ptac inducible promoter previously used for papillation assay (Figure 3B).
• Flow cytometry analysis was performed on 100,000 cells with a Beckman 362 Coulter Astrios EQ and data analysed using Weasel software v3.0.2.

Figures

Table 2: List of constitutive promoters.

Figure 1. Characterization of the papillation assay using a strong inducible promoter

Figure 3. Characterization of the set of constitutive promoters.

Full text

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A series of constitutive expression vectors to accurately measure the rate of DNA
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transposition and correct for auto-inhibition
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Michael Tellier* and Ronald Chalmers*
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School of Life Sciences, University of Nottingham, Queen’s Medical Centre, Nottingham,
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NG7 2UH, UK
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* Correspondence: michael.tellier@path.ox.ac.uk; ronald.chalmers@nottingham.ac.uk
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Present Address: Michael Tellier, Sir William Dunn School of Pathology, University of
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Oxford, Oxford, OX1 3RF, UK
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.CC-BY-NC-ND 4.0 International licensea
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Abstract
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Background
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Transposable elements (TEs) form a diverse group of DNA sequences encoding functions
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for their own mobility. This ability has been exploited as a powerful tool for molecular biology
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and genomics techniques. However, their use is sometimes limited because their activity is
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auto-regulated to allow them to cohabit within their hosts without causing excessive genomic
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damage. To overcome these limitations, it is important to develop efficient and simple
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screening assays for hyperactive transposases.
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Results
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To widen the range of transposase expression normally accessible with inducible promoters,
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we have constructed a set of vectors based on constitutive promoters of different strengths.
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We characterized and validated our expression vectors with Hsmar1, a member of the
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mariner transposon family. We observed the highest rate of transposition with the weakest
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promoters. We went on to investigate the effects of mutations in the Hsmar1 transposase
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dimer interface and of covalently linking two transposase monomers in a single-chain dimer.
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We also tested the severity of mutations in the lineage leading to the human SETMAR gene,
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in which one copy of the Hsmar1 transposase has contributed a domain.
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Conclusions
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We generated a set of vectors to provide a wide range of transposase expression which will
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be useful for screening libraries of transposase mutants. We also found that mutations in the
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Hsmar1 dimer interface provides resistance to overproduction inhibition in bacteria, which
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could be valuable for improving bacterial transposon mutagenesis techniques.
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
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Keywords
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Papillation assay, Hsmar1, overproduction inhibition, SETMAR, transposase, transposable
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elements.
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.CC-BY-NC-ND 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted October 18, 2019. ; https://doi.org/10.1101/423012doi: bioRxiv preprint

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Background
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Transposable elements (TEs) are DNA sequences encoding their own ability to move in a
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genome from one place to another. They are found in virtually all organisms and are
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particularly present in eukaryotes where they can represent a high percentage of the
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genome (1-3). Originally described as selfish elements since they were considered parasites
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which use the host for propagation but do not provide any particular advantage, TEs have
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now been shown to be important drivers of genome evolution (4, 5). Indeed, TEs can provide
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novel transcription factor binding sites, promoters, exons or poly(A) sites and can also be co-
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opted as microRNAs or long intergenic RNAs (6-8). TEs are a diverse group of DNA
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sequences using a wide range of mechanisms to transpose within their hosts. One particular
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mechanism prevalent in eukaryotes, and used by the mariner family, is known as “cut-and-
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paste” transposition (9). Over the past several years, our group and others have described
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the mechanisms regulating the transposition rate of different mariner transposons, such as
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Himar1, Hsmar1 or Mos1 (10-15). In Hsmar1, a regulatory mechanism was first recognized
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because of the phenomenon of overproduction inhibition (OPI) (16). The mechanism of OPI
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was eventually explained by the realization that double occupancy of the transposon ends
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with transposase dimers blocks assembly of the transpososome (12). Thus, OPI curbs
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Hsmar1 transposition rate to avoid damaging the host genome by excessive transposition
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(12).
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However, OPI represents a limitation in the development of hyperactive transposases, which
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would facilitate transposon mutagenesis. Several approaches such as modifying the binding
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kinetics of the transposase to the inverted terminal repeat (ITR) or the monomer-dimer
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equilibrium can be used to overcome OPI. Indeed, we and others previously showed that
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most mutations in the conserved motif, WVPHEL, in Himar1 and Hsmar1, located at the
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subunit interface, result in hyperactive transposases but at the cost of producing non-
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productive DNA double-strand breaks and therefore DNA damage (17, 18).
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.CC-BY-NC-ND 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted October 18, 2019. ; https://doi.org/10.1101/423012doi: bioRxiv preprint

5
To facilitate the isolation of suitable transposase mutants, the papillation assay was
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developed as an efficient screening procedure (Supplementary Figure 1) (19, 20). This
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assay is based on a promoter-less lacZ gene flanked by transposon ends. This reporter is
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integrated in a silent region of the genome of Escherichia coli. The transposase gene is
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provided in trans on a plasmid to simplify mutagenesis and library handling. Transposition
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events into an expressed ORF give rise to lacZ gene fusion proteins. When this happens
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within a colony growing on an X-gal indicator plate, it converts the cell to a lac+ phenotype,
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which allows the outgrowth of blue microcolonies (papillae) on a background of white cells.
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The transposition rate is estimated by the number of papillae per colony and by the rate of
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their appearance.
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A limitation of the papillation assay is that it generally employs a transposase gene whose
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expression is under the control of an inducible promoter which cannot be finely regulated.
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We have constructed a set of vectors maintained in single copy or as five copies per cell
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which carry various constitutive promoters in the absence or presence of a ribosome binding
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site (RBS). This set of vectors allows transposase expression across a wide range of
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expression levels facilitating the screening of hyperactive and/or OPI-resistant transposases.
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We used this set of vectors to compare an Hsmar1 transposase monomer to a single-chain
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dimer and to test for hyperactivity and OPI-resistance several Hsmar1 transposase mutants.
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We found that one Hsmar1 mutant in the dimer interface, R141L, is resistant to OPI in E. coli.
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Results and Discussion
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Characterization of the papillation assay using a strong inducible promoter
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The papillation assay provides a visual assessment of the transposition rate, which can be
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determined from the rate of papillae appearance and their number per colony (19). The
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transposition rate is dependent on the concentration and activity of the transposase (12). We
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defined the transposition rate as the average number of papillae per colony after five days of
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.CC-BY-NC-ND 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted October 18, 2019. ; https://doi.org/10.1101/423012doi: bioRxiv preprint