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• Wong, Che Fai Alex; Zhang, Shizhe; Tietze, Lisa; Dahiya, Gurvinder Singh & Lale, Rahmi (2025). Engineering artificial 5′ regulatory sequences for thermostable protein expression in the extremophile Thermus thermophilus. Synthetic Biology. 10(1). doi: 10.1093/synbio/ysaf016. Full text in Research Archive
• Totlani, Kartik; Khan, Essa Ahsan; Ahmed, Husnain; Lale, Rahmi & Stokke, Bjorn Torger (2025). Integrated microdroplet workflow for high-throughput cell-free transcription in double emulsion picoreactors. BioRxiv. ISSN 0362-4331. doi: 10.1101/2025.11.13.688266. Full text in Research Archive Show summary

  Single-molecule nucleic acid assays offer a powerful route to understand gene regulation. To enable rapid and precise characterization of regulatory sequences such as promoters within a pool of synthetic 5' regulatory sequences, we present an integrated microdroplet workflow for high-throughput cell-free transcription in double emulsion (DE) picoreactors. For this, we utilize the T7 promoter as a positive control and the Mango III RNA aptamer for fluorescent readout of transcription in an in vitro setup. First, DNA templates are compartmentalized into picolitre droplets along with rolling circle amplification reagents and incubated off‐chip. The resulting emulsion is then reinjected into a second device that integrates three operations on a single chip : (i) emulsion reinjection, (ii) electric-field-mediated StepInjection of IVT reagents and (iii) downstream water-in-oil-in-water (W/O/W) double emulsification. Post IVT incubation, the fluorescent DE droplets are selectively isolated by FACS, enabling sorting as well as the classification of droplets on the basis of their fluorescent intensities. We characterize the workflow with proof-of-principle experiments with three input DNA concentrations that yield distinct fractions of fluorescent DEs and also demonstrate intensity-based binning and indexed sorting of droplets into defined wells of a microtiter plate. This workflow establishes a practical route to high-throughput, single-template transcription assays in FACS compatible double emulsions and provides a foundation for future screening of synthetic regulatory libraries and promoter variants with fluorescence encoded readouts.

• Øvreeide, Ingrid Haga; Sturdy, Jacob Trent; Szydlak, Renata; Hines, Thomas Gulvik; Ahmed, Husnain & Totlani, Kartik Arun [Show all 11 contributors for this article] (2025). Microfluidic constriction-based mechanoprofiling of alginate microgels and bladder cancer cells. Polymer. ISSN 0032-3861. 337. doi: 10.1016/j.polymer.2025.128958. Full text in Research Archive
• Khan, Essa Ahsan; Rückert-Reed, Christian; Dahiya, Gurvinder Singh; Tietze, Lisa; Fages-Lartaud, Maxime René & Busche, Tobias [Show all 9 contributors for this article] (2025). High-resolution mapping of sigma factor DNA-binding sequences using artificial promoters, RNA aptamers, and deep sequencing. Nucleic Acids Research (NAR). ISSN 0305-1048. 53(7). doi: 10.1093/nar/gkaf306. Full text in Research Archive Show summary

  Abstract The variable sigma (σ) subunit of the bacterial RNA polymerase holoenzyme determines promoter specificity and facilitates open complex formation during transcription initiation. Understanding σ-factor binding sequences is therefore crucial for deciphering bacterial gene regulation. Here, we present a data-driven high-throughput approach that utilizes an extensive library of 1.54 million DNA templates providing artificial promoters and 5′ untranslated region sequences for σ-factor DNA-binding motif discovery. This method combines the generation of extensive DNA libraries, in vitro transcription, RNA aptamer, and deep DNA and RNA sequencing. It allows direct assessment of promoter activity, identification of transcription start sites, and quantification of promoter strength based on mRNA production levels. We applied this approach to map σ54 DNA-binding sequences in Pseudomonas putida. Deep sequencing of the enriched RNA pool revealed 64 966 distinct σ54 binding motifs, significantly expanding the known repertoire. This data-driven approach surpasses traditional methods by directly evaluating promoter function and avoiding selection bias based solely on binding affinity. This comprehensive dataset enhances our understanding of σ-factor binding sequences and their regulatory roles, opening avenues for new research in biology and biotechnology.

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• Kjellevold-Skallerud, Embla; Pettersen, Maia Lapidus; Totlani, Kartik Arun; Prot, Victorien Emile & Stokke, Bjørn Torger (2025). Viscoelastic relaxation of monodisperse Ca-alginate microgel beads via constriction in microfluidic devices. Full text in Research Archive
• Khan, Essa Ahsan; Rückert-Reed, Christian; Dahiya, Gurvinder Singh; Tietze, Lisa; Fages-Lartaud, Maxime René & Busche, Tobias [Show all 9 contributors for this article] (2024). High-resolution mapping of Sigma Factor DNA Binding Sequences using Artificial Promoters, RNA aptamers and Deep Sequencing in Pseudomonas putida. Full text in Research Archive
• Lale, Rahmi (2024). High-resolution mapping of Sigma Factor DNA Binding Sequences using Artificial Promoters, RNA aptamers and Deep Sequencing. Full text in Research Archive
• Lale, Rahmi (2024). Leveraging AI for Context-Aware and Predictable DNA Design in Synthetic Biology. Full text in Research Archive
• Ahmed, Husnain; Khan, Essa Ahsan; Tietze, Lisa; Fages-Lartaud, Maxime René; Totlani, Kartik & Bjørkøy, Astrid [Show all 8 contributors for this article] (2022). Unravelling the sigma factor specific promoter binding in bacterial in-vitro transcription using droplet microfluidics. Full text in Research Archive
• Ahmed, Husnain; Khan, Essa Ahsan; Tietze, Lisa; Fages-Lartaud, Maxime René; Totlani, Kartik & Bjørkøy, Astrid [Show all 8 contributors for this article] (2022). Matchmaking inside microdroplets: unravelling the sigma factor specific promoter binding in bacterial in-vitro transcription using droplet microfluidics. Full text in Research Archive

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