Tunable transcription factor library for robust quantification of regulatory properties in
            <i>Escherichia coli</i>

Parisutham, Vinuselvi; Chhabra, Shivani; Ali, M. Zulfikar; Brewster, Robert C.

doi:10.15252/msb.202110843

Cited by 14 publications

(21 citation statements)

References 70 publications

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“…Previous DAP-seq binding analysis using purified TFs and genomic DNA fragments identified MetR binding sites at metF and folE promoters 42 , but the in vivo regulatory responses have never been tested. We further verified these regulatory responses using a MetR knockdown strain from the tunable TF library 34 (Fig. 4c ).…”

Section: Resultsmentioning

confidence: 85%

“…showed only weak perturbation effects (i.e., less than 30% promoter activity change) under our growth conditions in minimal glucose medium. To further validate these findings, we selected seven TF genes and measured their promoter activities across a wide range of TF concentrations using a tunable E. coli TF library 34 , in which each endogenous TF is replaced by an inducible TF-mCherry fusion (Supplementary Fig. 8 ).…”

Section: Resultsmentioning

confidence: 99%

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Genome-wide promoter responses to CRISPR perturbations of regulators reveal regulatory networks in Escherichia coli

Han,

Li,

Filko

et al. 2023

Nat Commun

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Elucidating genome-scale regulatory networks requires a comprehensive collection of gene expression profiles, yet measuring gene expression responses for every transcription factor (TF)-gene pair in living prokaryotic cells remains challenging. Here, we develop pooled promoter responses to TF perturbation sequencing (PPTP-seq) via CRISPR interference to address this challenge. Using PPTP-seq, we systematically measure the activity of 1372 Escherichia coli promoters under single knockdown of 183 TF genes, illustrating more than 200,000 possible TF-gene responses in one experiment. We perform PPTP-seq for E. coli growing in three different media. The PPTP-seq data reveal robust steady-state promoter activities under most single TF knockdown conditions. PPTP-seq also enables identifications of, to the best of our knowledge, previously unknown TF autoregulatory responses and complex transcriptional control on one-carbon metabolism. We further find context-dependent promoter regulation by multiple TFs whose relative binding strengths determined promoter activities. Additionally, PPTP-seq reveals different promoter responses in different growth media, suggesting condition-specific gene regulation. Overall, PPTP-seq provides a powerful method to examine genome-wide transcriptional regulatory networks and can be potentially expanded to reveal gene expression responses to other genetic elements.

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Section: Resultsmentioning

confidence: 85%

Section: Resultsmentioning

confidence: 99%

Genome-wide promoter responses to CRISPR perturbations of regulators reveal regulatory networks in Escherichia coli

Han,

Li,

Filko

et al. 2023

Nat Commun

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“…For example, combining our GR reporters with strains from the KEIO collection (Baba et al , 2006) and the ASKA library (Kitagawa et al , 2005) should allow assessing how much a given gene influences the GRs. Similarly, a ‘Tunable TF library’(Parisutham et al , 2022) was recently produced to synthetically tune the concentration of most TFs. However, it cannot monitor natural production rates.…”

Section: Discussionmentioning

confidence: 99%

“…We used E. coli K-12 MG1655 cells (WT strain), as it is also used in (Zaslaver et al , 2006; Taniguchi et al , 2010; Parisutham et al , 2022). Chemically competent (CC) E. coli K-12 MG1655 cells were prepared for plasmid transformation.…”

Section: Methodsmentioning

confidence: 99%

A library of reporters of the global regulators of gene expression ofEscherichia coli

Dash,

Jagadeesan,

Baptista

et al. 2023

Preprint

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SUMMARYThe topology of the transcription factor network (TFN) ofE. coliis far from uniform, with 22 global regulator (GR) proteins controlling one-third of all genes. So far, their production rates cannot be tracked by comparable fluorescent proteins. We developed a library of fluorescent reporters for 16 GRs for this purpose. Each consists of a single-copy plasmid coding for GFP fused to the full-length copy of the native promoter. We tracked their activity in exponential and stationary growth, as well as under weak and strong stresses. We show that the reporters have high sensitivity and specificity to all stresses tested and detect single-cell variability in transcription rates. Given the influence of GRs on the TFN, we expect that the new library will contribute to dissecting global transcriptional stress-response programs ofE. coli. Moreover, the library can be invaluable in bioindustrial applications that tune those programs to, instead of cell growth, favor productivity while reducing energy consumption.Abstract Figure

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“…mini‐Tn5) and contained ~1.4 × 10 5 independent clones (Scholz et al., 2019 ). Other integrated libraries are limited to a maximum of 3 × 10 4 clones (Biggs et al., 2020 ; Cowie et al., 2006 ; Elmore et al., 2017 ; Parisutham et al., 2022 ; Saleski et al., 2021 ). Developing efficient systems that can integrate large gene libraries would facilitate more effective screening of combinatorial libraries and the implementation of continuous selection processes in synthetic evolution (Simon et al., 2019 ).…”

Section: Introductionmentioning

confidence: 99%

Massive integration of large gene libraries in the chromosome of Escherichia coli

Cerdán,

Álvarez,

Fernández

2023

Microbial Biotechnology

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Large gene libraries are frequently created in Escherichia coli plasmids, which can induce cell toxicity and expression instability due to the high gene dosage. To address these limitations, gene libraries can be integrated in a single copy into the bacterial chromosome. Here, we describe an efficient system for the massive integration (MAIN) of large gene libraries in the E. coli chromosome that generates in‐frame gene fusions that are expressed stably. MAIN uses a thermosensitive integrative plasmid that is linearized in vivo to promote extensive integration of the gene library via homologous recombination. Positive and negative selections efficiently remove bacteria lacking gene integration in the target site. We tested MAIN with a library of 107 VHH genes that encode nanobodies (Nbs). The integration of VHH genes into a custom target locus of the E. coli chromosome enabled stable expression and surface display of the Nbs. Next‐generation DNA sequencing confirmed that MAIN preserved the diversity of the gene library after integration. Finally, we screened the integrated library to select Nbs that bind a specific antigen using magnetic and fluorescence‐activated cell sorting. This allowed us to identify Nbs binding the epidermal growth factor receptor that were not previously isolated in a similar screening of a multicopy plasmid library. Our results demonstrate that MAIN enables large gene library integration into the E. coli chromosome, creating stably expressed in‐frame fusions for functional screening.

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Tunable transcription factor library for robust quantification of regulatory properties in Escherichia coli

Cited by 14 publications

References 70 publications

Genome-wide promoter responses to CRISPR perturbations of regulators reveal regulatory networks in Escherichia coli

Genome-wide promoter responses to CRISPR perturbations of regulators reveal regulatory networks in Escherichia coli

A library of reporters of the global regulators of gene expression ofEscherichia coli

Massive integration of large gene libraries in the chromosome of Escherichia coli

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