Sourcing Phage-Encoded Terminators Using ONT-cappable-seq for SynBio Applications in

Overview

Journal ACS Synth Biol

Publisher American Chemical Society

Specialties Biotechnology
Molecular Biology

Date 2023 Apr 24

PMID 37092882

Authors

Eveline-Marie Lammens

Leena Putzeys

Maarten Boon

Rob Lavigne

Affiliations

Soon will be listed here.

Abstract

Efficient transcriptional terminators are essential for the performance of genetic circuitry in microbial SynBio hosts. In recent years, several libraries of characterized strong terminators have become available for model organisms such as . Conversely, terminator libraries for nonmodel species remain scarce, and individual terminators are often ported over from model systems, leading to unpredictable performance in their new hosts. In this work, we mined the genomes of infecting phages LUZ7 and LUZ100 for transcriptional terminators utilizing the full-length RNA sequencing technique "ONT-cappable-seq" and validated these terminators in three Gram-negative hosts using a terminator trap assay. Based on these results, we present nine terminators for , , and , which outperform current reference terminators. Among these, terminator LUZ7 T50 displays potent bidirectional activity. These data further support that bacteriophages, as evolutionary-adapted natural predators of the targeted bacteria, provide a valuable source of microbial SynBio parts.

Citing Articles

Engineering a phi15-based expression system for stringent gene expression in Pseudomonas putida.

Lammens E, Volke D, Kerremans A, Aerts Y, Boon M, Nikel P Commun Biol. 2025; 8(1):171.

PMID: 39905116 PMC: 11794488. DOI: 10.1038/s42003-025-07508-y.

Assessing the transcriptional landscape of Pseudomonas phage 201ϕ2-1: Uncovering the small regulatory details of a giant phage.

Poppeliers J, Focquet M, Boon M, De Mey M, Thomas J, Lavigne R Microb Biotechnol. 2024; 17(10):e70037.

PMID: 39460739 PMC: 11512511. DOI: 10.1111/1751-7915.70037.

Obtaining Detailed Phage Transcriptomes Using ONT-Cappable-Seq.

Putzeys L, Intizar D, Lavigne R, Boon M Methods Mol Biol. 2024; 2793:207-235.

PMID: 38526733 DOI: 10.1007/978-1-0716-3798-2_14.

Meeting Report of the Second Symposium of the Belgian Society for Viruses of Microbes and Launch of the Phage Valley.

Desmecht S, Latka A, Ceyssens P, Garcia-Pino A, Gillis A, Lavigne R Viruses. 2024; 16(2).

PMID: 38400074 PMC: 10891784. DOI: 10.3390/v16020299.

Exploring the transcriptional landscape of phage-host interactions using novel high-throughput approaches.

Putzeys L, Wicke L, Brandao A, Boon M, Pires D, Azeredo J Curr Opin Microbiol. 2024; 77:102419.

PMID: 38271748 PMC: 10884466. DOI: 10.1016/j.mib.2023.102419.

References

Mairhofer J, Wittwer A, Cserjan-Puschmann M, Striedner G . Preventing T7 RNA polymerase read-through transcription-A synthetic termination signal capable of improving bioprocess stability. ACS Synth Biol. 2014; 4(3):265-73. DOI: 10.1021/sb5000115. View

Lorenz R, Bernhart S, Honer Zu Siederdissen C, Tafer H, Flamm C, Stadler P . ViennaRNA Package 2.0. Algorithms Mol Biol. 2011; 6:26. PMC: 3319429. DOI: 10.1186/1748-7188-6-26. View

Choe D, Kim K, Kang M, Lee S, Cho S, Palsson B . Synthetic 3'-UTR valves for optimal metabolic flux control in Escherichia coli. Nucleic Acids Res. 2022; 50(7):4171-4186. PMC: 9023263. DOI: 10.1093/nar/gkac206. View

Wang L, Watters J, Ju X, Lu G, Liu S . Head-on and co-directional RNA polymerase collisions orchestrate bidirectional transcription termination. Mol Cell. 2023; 83(7):1153-1164.e4. PMC: 10081963. DOI: 10.1016/j.molcel.2023.02.017. View

Grunberger F, Ferreira-Cerca S, Grohmann D . Nanopore sequencing of RNA and cDNA molecules in . RNA. 2021; 28(3):400-417. PMC: 8848933. DOI: 10.1261/rna.078937.121. View

Lammens E, Boon M, Grimon D, Briers Y, Lavigne R . SEVAtile: a standardised DNA assembly method optimised for Pseudomonas. Microb Biotechnol. 2021; 15(1):370-386. PMC: 8719830. DOI: 10.1111/1751-7915.13922. View

He Z, Duan Y, Zhai W, Zhang X, Shi J, Zhang X . Evaluating Terminator Strength Based on Differentiating Effects on Transcription and Translation. Chembiochem. 2020; 21(14):2067-2072. DOI: 10.1002/cbic.202000068. View

Putzeys L, Poppeliers J, Boon M, Lood C, Vallino M, Lavigne R . Transcriptomics-Driven Characterization of LUZ100, a T7-like Pseudomonas Phage with Temperate Features. mSystems. 2023; 8(2):e0118922. PMC: 10134795. DOI: 10.1128/msystems.01189-22. View

Lammens E, Nikel P, Lavigne R . Exploring the synthetic biology potential of bacteriophages for engineering non-model bacteria. Nat Commun. 2020; 11(1):5294. PMC: 7576135. DOI: 10.1038/s41467-020-19124-x. View

10.

Naville M, Ghuillot-Gaudeffroy A, Marchais A, Gautheret D . ARNold: a web tool for the prediction of Rho-independent transcription terminators. RNA Biol. 2011; 8(1):11-3. DOI: 10.4161/rna.8.1.13346. View

11.

Yan B, Boitano M, Clark T, Ettwiller L . SMRT-Cappable-seq reveals complex operon variants in bacteria. Nat Commun. 2018; 9(1):3676. PMC: 6131387. DOI: 10.1038/s41467-018-05997-6. View

12.

de Felippes F, McHale M, Doran R, Roden S, Eamens A, Finnegan E . The key role of terminators on the expression and post-transcriptional gene silencing of transgenes. Plant J. 2020; 104(1):96-112. DOI: 10.1111/tpj.14907. View

13.

Nielsen A, Der B, Shin J, Vaidyanathan P, Paralanov V, Strychalski E . Genetic circuit design automation. Science. 2016; 352(6281):aac7341. DOI: 10.1126/science.aac7341. View

14.

Cambray G, Guimaraes J, Mutalik V, Lam C, Mai Q, Thimmaiah T . Measurement and modeling of intrinsic transcription terminators. Nucleic Acids Res. 2013; 41(9):5139-48. PMC: 3643576. DOI: 10.1093/nar/gkt163. View

15.

Nielsen A, Segall-Shapiro T, Voigt C . Advances in genetic circuit design: novel biochemistries, deep part mining, and precision gene expression. Curr Opin Chem Biol. 2013; 17(6):878-92. DOI: 10.1016/j.cbpa.2013.10.003. View

16.

Ju X, Li D, Liu S . Full-length RNA profiling reveals pervasive bidirectional transcription terminators in bacteria. Nat Microbiol. 2019; 4(11):1907-1918. PMC: 6814526. DOI: 10.1038/s41564-019-0500-z. View

17.

Hudson A, Wieden H . Rapid generation of sequence-diverse terminator libraries and their parameterization using quantitative Term-Seq. Synth Biol (Oxf). 2020; 4(1):ysz026. PMC: 7445774. DOI: 10.1093/synbio/ysz026. View

18.

Roberts J . Mechanisms of Bacterial Transcription Termination. J Mol Biol. 2019; 431(20):4030-4039. DOI: 10.1016/j.jmb.2019.04.003. View

19.

Putzeys L, Boon M, Lammens E, Kuznedelov K, Severinov K, Lavigne R . Development of ONT-cappable-seq to unravel the transcriptional landscape of phages. Comput Struct Biotechnol J. 2022; 20:2624-2638. PMC: 9163698. DOI: 10.1016/j.csbj.2022.05.034. View

20.

Yamanishi M, Ito Y, Kintaka R, Imamura C, Katahira S, Ikeuchi A . A genome-wide activity assessment of terminator regions in Saccharomyces cerevisiae provides a ″terminatome″ toolbox. ACS Synth Biol. 2013; 2(6):337-47. DOI: 10.1021/sb300116y. View