An improved Escherichia coli strain to host gene regulatory networks involving both the AraC and LacI inducible transcription factors

Kogenaru, Manjunatha; Tans, Sander J.

doi:10.1186/1754-1611-8-2

Cited by 36 publications

(38 citation statements)

References 9 publications

(13 reference statements)

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“…Ligation (“blue” and “green” regulatory regions) and Gibson assembly reaction products (“red” regulatory region) were transformed into electrocompetent MK01 cells (Kogenaru and Tans, 2014) that carried already the two other plasmids necessary to complete the synthetic circuit. Transformants were plated out on SM-agar plates.…”

Section: Methodsmentioning

confidence: 99%

“…The extracted plasmid libraries were mixed with plasmids containing no mutations in a ratio of 70:30 to generate mutant circuits that have mutations in two or three genes. The resulting plasmid mix was transformed into electrocompetent MK01 cells (Kogenaru and Tans, 2014). Circuits that had no mutations or only mutations in one gene were not considered in the analysis.…”

Section: Methodsmentioning

confidence: 99%

“…For SI Fig. 5b the mutated plasmid coding for the “blue” gene was transformed together with the WT plasmids for the “green” and “red” genes into electrocompetent MK01 cells (Kogenaru and Tans, 2014). For SI Fig.…”

Section: Methodsmentioning

confidence: 99%

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The mechanisms of gene regulatory networks constrain evolution: A lesson from synthetic circuits

Schaerli

Jiménez

et al. 2017

Preprint

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Phenotypic variation is the raw material of adaptive Darwinian evolution. The phenotypic variation found in organismal development is biased towards certain phenotypes, but the molecular mechanisms behind such restrictions are still poorly understood. Gene regulatory networks have been proposed as one cause of constrained phenotypic variation. However, most of the evidence for this is theoretical rather than experimental. Here, we study evolutionary biases in two synthetic gene regulatory circuits expressed in E. coli that produce a gene expression stripe - a pivotal pattern in embryonic development. The two parental circuits produce the same phenotype, but create it through different regulatory mechanisms. We show that mutations cause distinct novel phenotypes in the two networks and use a combination of experimental measurements, mathematical modelling and DNA sequencing to understand why mutations bring forth only some but not other novel gene expression phenotypes. Our results reveal that the regulatory mechanisms of networks restrict the possible phenotypic variation upon mutation. Consequently, seemingly equivalent networks can indeed be distinct in how they constrain the outcome of further evolution.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The mechanisms of gene regulatory networks constrain evolution: A lesson from synthetic circuits

Schaerli

Jiménez

et al. 2017

Preprint

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“…Microbe strain and growth conditions E.coli BW27783 MK01 strain (kindly provided by the M.Isalan lab), modified to homogenously express arabinose-induced genes (Kogenaru and Tans, 2014) was used to express the mutant library. A single colony of the E.coli BW27783 MK01 strain was picked Luria-Bertani (LB) agar plate, grown overnight at LB liquid medium supplemented with chloramphenicol to 14μg/ml concentration at 37°C.…”

Section: Experimental Model and Subject Detailsmentioning

confidence: 99%

“…We chose the E.coli BW27783 MK01 strain (kindly provided by the Isalan lab) (Kogenaru and Tans, 2014), modified to homogenously express arabinose-induced genes, to express the mutant library. A single chloramphenicol-resistant colony of was picked into 4ml LB medium with 2.8μl of 20mg/ml chloramphenicol and let grow for 3.5 hours at 37°C.…”

Section: Making Highly Efficient Electro-competent Cellsmentioning

confidence: 99%

Changes in gene expression shift and switch genetic interactions

Lalić

Baeza-Centurion

et al. 2019

Preprint

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One sentence Summary:Deep mutagenesis of the lambda repressor reveals that changes in gene expression will alter the strength and direction of genetic interactions between mutations in many genes. SummaryAn important goal in disease genetics and evolutionary biology is to understand how mutations combine to alter phenotypes and fitness. Non-additive interactions between mutations occur extensively and change across conditions, cell types, and species, making genetic prediction a difficult challenge. To understand the reasons for this, we reduced the problem to a minimal system where we combined mutations in a single protein performing a single function (a transcriptional repressor inhibiting a target gene). Even in this minimal system, a change in gene expression altered both the strength and type of genetic interactions. These seemingly complicated changes could, however, be predicted by a mathematical model that propagates the effects of mutations on protein folding to the cellular phenotype. We show that similar changes will be observed for many genes.These results provide fundamental insights into genotype-phenotype maps and illustrate how changes in genetic interactions can be predicted using hierarchical mechanistic models. Highlights• Deep mutagenesis of the lambda repressor at two expression levels reveals extensive changes in mutational effects and genetic interactions • Genetic interactions can switch from positive (suppressive) to negative (enhancing) as the expression of a gene changes • A mathematical model that propagates the effects of mutations on protein folding to the cellular phenotype accurately predicts changes in mutational effects and interactions• Changes in expression will alter mutational effects and interactions for many genes • For some genes, perfect mechanistic models will never be able to predict how mutations of known effect combine without measurements of intermediate phenotypes Schueller Foundation, Agencia de Gestio d'Ajuts Universitaris i de Recerca (AGAUR, 2017 SGR 1322, and the CERCA Program/Generalitat de Catalunya. X. Li was supported in part by a fellowship from the Ramón Areces Foundation.

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Synthetic Gene Circuits Combining CRISPR Interference and CRISPR Activation in E. coli: Importance of Equal Guide RNA Binding Affinities to Avoid Context-Dependent Effects

Barbier,

Kusumawardhani,

Chauhan

et al. 2023

ACS Synth. Biol.

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Gene expression control based on clustered regularly interspaced short palindromic repeats (CRISPR) has emerged as a powerful approach for constructing synthetic gene circuits. While the use of CRISPR interference (CRISPRi) is already well-established in prokaryotic circuits, CRISPR activation (CRISPRa) is less mature, and a combination of the two in the same circuits is only just emerging. Here, we report that combining CRISPRi with SoxS-based CRISPRa in Escherichia coli can lead to context-dependent effects due to different affinities in the formation of CRISPRa and CRISPRi complexes, resulting in loss of predictable behavior. We show that this effect can be avoided by using the same scaffold guide RNA structure for both complexes.

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An improved Escherichia coli strain to host gene regulatory networks involving both the AraC and LacI inducible transcription factors

Cited by 36 publications

References 9 publications

The mechanisms of gene regulatory networks constrain evolution: A lesson from synthetic circuits

The mechanisms of gene regulatory networks constrain evolution: A lesson from synthetic circuits

Changes in gene expression shift and switch genetic interactions

Synthetic Gene Circuits Combining CRISPR Interference and CRISPR Activation in E. coli: Importance of Equal Guide RNA Binding Affinities to Avoid Context-Dependent Effects

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