Refactoring of a synthetic raspberry ketone pathway with EcoFlex

Abstract Background A key focus of synthetic biology is to develop microbial or cell-free based biobased routes to value-added chemicals such as fragrances. Originally, we developed the EcoFlex system, a Golden Gate toolkit, to study genes/pathways flexibly using Escherichia coli heterologous expression. In this current work, we sought to use EcoFlex to optimise a synthetic raspberry ketone biosynthetic pathway. Raspberry ketone is a high-value (~ £20,000 kg−1) fine chemical farmed from raspberry (Rubeus rubrum) fruit. Results By applying a synthetic biology led design-build-test-learn cycle approach, we refactor the raspberry ketone pathway from a low level of productivity (0.2 mg/L), to achieve a 65-fold (12.9 mg/L) improvement in production. We perform this optimisation at the prototype level (using microtiter plate cultures) with E. coli DH10β, as a routine cloning host. The use of E. coli DH10β facilitates the Golden Gate cloning process for the screening of combinatorial libraries. In addition, we also newly establish a novel colour-based phenotypic screen to identify productive clones quickly from solid/liquid culture. Conclusions Our findings provide a stable raspberry ketone pathway that relies upon a natural feedstock (L-tyrosine) and uses only constitutive promoters to control gene expression. In conclusion we demonstrate the capability of EcoFlex for fine-tuning a model fine chemical pathway and provide a range of newly characterised promoter tools gene expression in E. coli.

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CRIMoClo plasmids for modular assembly and orthogonal chromosomal integration of synthetic circuits in Escherichia coli

Journal of Biological Engineering ◽

10.1186/s13036-019-0218-8 ◽

2019 ◽

Vol 13 (1) ◽

Cited By ~ 1

Author(s):

Stefano Vecchione ◽

Georg Fritz

Keyword(s):

Escherichia Coli ◽

Synthetic Biology ◽

Integrated Circuits ◽

High Efficiency ◽

Genetic Circuit ◽

Dna Assembly ◽

Chromosomal Integration ◽

Golden Gate ◽

Genetic Circuits ◽

E Coli

Abstract Background Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. Results By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. Conclusions The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

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Dual UTR-A novel 5′ untranslated region design for synthetic biology applications

Synthetic Biology ◽

10.1093/synbio/ysaa006 ◽

2020 ◽

Vol 5 (1) ◽

Author(s):

Simone Balzer Le ◽

Ingerid Onsager ◽

Jon Andreas Lorentzen ◽

Rahmi Lale

Keyword(s):

Gene Expression ◽

Synthetic Biology ◽

De Novo ◽

Expression Profiles ◽

Regulation Of Gene Expression ◽

Design Concept ◽

Fine Tuning ◽

The Novel ◽

Wide Range ◽

Application Potential

Abstract Bacterial 5′ untranslated regions of mRNA (UTR) involve in a complex regulation of gene expression; however, the exact sequence features contributing to gene regulation are not yet fully understood. In this study, we report the design of a novel 5′ UTR, dual UTR, utilizing the transcriptional and translational characteristics of 5′ UTRs in a single expression cassette. The dual UTR consists of two 5′ UTRs, each separately leading to either increase in transcription or translation of the reporter, that are separated by a spacer region, enabling de novo translation initiation. We rationally create dual UTRs with a wide range of expression profiles and demonstrate the functionality of the novel design concept in Escherichia coli and Pseudomonas putida using different promoter systems and coding sequences. Overall, we demonstrate the application potential of dual UTR design concept in various synthetic biology applications ranging from fine-tuning of gene expression to maximization of protein production.

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UNIGEMS: plasmids and parts to facilitate teaching on assembly, gene expression control and logic in E. coli

10.1101/2021.06.20.449138 ◽

2021 ◽

Author(s):

Alex Siddall ◽

Abbie Ann Williams ◽

Jason Sanders ◽

Jai A Denton ◽

Dean Madden ◽

...

Keyword(s):

Gene Expression ◽

Synthetic Biology ◽

Logic Gate ◽

Undergraduate Teaching ◽

Gibson Assembly ◽

E Coli ◽

Expression Control ◽

Gene Expression Control ◽

Plasmid Backbone ◽

High Copy Plasmid

Synthetic biology is as an excellent vehicle for education, as it enables creativecombination of engineering and molecular biology approaches for quantitative charac-terisations of the assembled constructs. However, there is a limited number of resourcesavailable for such applications in the educational context, where straightforward setup, easily measurable phenotypes and extensibility are of particular importance. To expandthe availability of education-friendly resources to teach synthetic biology and geneticengineering, we developed Unigems, a set of 10 plasmids that enable out-of-the-boxinvestigations of principles of gene expression control, as well as more complex designs- a biological logic gate. The system uses a common high-copy plasmid backbone anda common set of primers to enable Gibson-assembly of PCR-generated or synthesisedparts into a target vector. It currently has two reporter genes with either two consti-tutive (high- or low-level) or two inducible (lactose- or arabinose-) promoters, as wellas a single-plasmid implementation of an AND logic gate. The Unigems system hasalready been employed in undergraduate teaching settings, during outreach events andfor training of iGEM teams. All plasmids have been deposited in Addgene.

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Robust gene expression control in human cells with a novel universal TetR aptamer splicing module

Nucleic Acids Research ◽

10.1093/nar/gkz753 ◽

2019 ◽

Vol 47 (20) ◽

pp. e132-e132 ◽

Cited By ~ 5

Author(s):

Adam A Mol ◽

Florian Groher ◽

Britta Schreiber ◽

Ciaran Rühmkorff ◽

Beatrix Suess

Keyword(s):

Gene Expression ◽

Synthetic Biology ◽

Dynamic Range ◽

Intron Retention ◽

Human Cells ◽

Fine Tuning ◽

Genomic Context ◽

Mammalian Cell Lines ◽

Gene Expression Control ◽

Wide Range

Abstract Fine-tuning of gene expression is desirable for a wide range of applications in synthetic biology. In this context, RNA regulatory devices provide a powerful and highly functional tool. We developed a versatile, robust and reversible device to control gene expression by splicing regulation in human cells using an aptamer that is recognized by the Tet repressor TetR. Upon insertion in proximity to the 5′ splice site, intron retention can be controlled via the binding of TetR to the aptamer. Although we were able to demonstrate regulation for different introns, the genomic context had a major impact on regulation. In consequence, we advanced the aptamer to develop a splice device. Our novel device contains the aptamer integrated into a context of exonic and intronic sequences that create and maintain an environment allowing a reliable and robust splicing event. The exon-born, additional amino acids will then be cleaved off by a self-cleaving peptide. This design allows portability of the splicing device, which we confirmed by demonstrating its functionality in different gene contexts. Intriguingly, our splicing device shows a high dynamic range and low basal activity, i.e. desirable features that often prove a major challenge when implementing synthetic biology in mammalian cell lines.

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Unprecedented High-Resolution View of Bacterial Operon Architecture Revealed by RNA Sequencing

mBio ◽

10.1128/mbio.01442-14 ◽

2014 ◽

Vol 5 (4) ◽

Cited By ~ 92

Author(s):

Tyrrell Conway ◽

James P. Creecy ◽

Scott M. Maddox ◽

Joe E. Grissom ◽

Trevor L. Conkle ◽

...

Keyword(s):

Gene Expression ◽

High Resolution ◽

Rna Sequencing ◽

Fine Tuning ◽

Logarithmic Phase ◽

Antisense Transcripts ◽

Single Nucleotide ◽

Content Type ◽

E Coli ◽

K 12

ABSTRACTWe analyzed the transcriptome ofEscherichia coliK-12 by strand-specific RNA sequencing at single-nucleotide resolution during steady-state (logarithmic-phase) growth and upon entry into stationary phase in glucose minimal medium. To generate high-resolution transcriptome maps, we developed an organizational schema which showed that in practice only three features are required to define operon architecture: the promoter, terminator, and deep RNA sequence read coverage. We precisely annotated 2,122 promoters and 1,774 terminators, defining 1,510 operons with an average of 1.98 genes per operon. Our analyses revealed an unprecedented view ofE. colioperon architecture. A large proportion (36%) of operons are complex with internal promoters or terminators that generate multiple transcription units. For 43% of operons, we observed differential expression of polycistronic genes, despite being in the same operons, indicating thatE. colioperon architecture allows fine-tuning of gene expression. We found that 276 of 370 convergent operons terminate inefficiently, generating complementary 3′ transcript ends which overlap on average by 286 nucleotides, and 136 of 388 divergent operons have promoters arranged such that their 5′ ends overlap on average by 168 nucleotides. We found 89 antisense transcripts of 397-nucleotide average length, 7 unannotated transcripts within intergenic regions, and 18 sense transcripts that completely overlap operons on the opposite strand. Of 519 overlapping transcripts, 75% correspond to sequences that are highly conserved inE. coli(>50 genomes). Our data extend recent studies showing unexpected transcriptome complexity in several bacteria and suggest that antisense RNA regulation is widespread.IMPORTANCEWe precisely mapped the 5′ and 3′ ends of RNA transcripts across theE. coliK-12 genome by using a single-nucleotide analytical approach. Our resulting high-resolution transcriptome maps show that ca. one-third ofE. colioperons are complex, with internal promoters and terminators generating multiple transcription units and allowing differential gene expression within these operons. We discovered extensive antisense transcription that results from more than 500 operons, which fully overlap or extensively overlap adjacent divergent or convergent operons. The genomic regions corresponding to these antisense transcripts are highly conserved inE. coli(includingShigellaspecies), although it remains to be proven whether or not they are functional. Our observations of features unearthed by single-nucleotide transcriptome mapping suggest that deeper layers of transcriptional regulation in bacteria are likely to be revealed in the future.

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BacilloFlex: A modular DNA assembly toolkit forBacillus subtilissynthetic biology

10.1101/185108 ◽

2017 ◽

Cited By ~ 1

Author(s):

Niels Wicke ◽

David Radford ◽

Valeria Verrone ◽

Anil Wipat ◽

Christopher E. French

Keyword(s):

Synthetic Biology ◽

Cellular Differentiation ◽

Genetic Manipulation ◽

Surface Display ◽

Dna Assembly ◽

Golden Gate ◽

E Coli ◽

Production Platform ◽

Bacterial Model ◽

Golden Gate Cloning

AbstractBacillus subtilisis a valuable industrial production platform for proteins, a bacterial model for cellular differentiation and its endospores have been proposed as a vehicle for vaccine delivery. As suchB. subtilisis a major synthetic biology chassis but, unlikeEscherichia coli, lacks a standardized toolbox for genetic manipulation. EcoFlex is a versatile modular DNA assembly toolkit forE. colisynthetic biology based on Golden Gate cloning. Here we introduce BacilloFlex, an extension of the EcoFlex assembly standard toB. subtilis. Transcription units flanked by sequences homologous to loci in theB. subtilisgenome were rapidly assembled by the EcoFlex standard and subsequently chromosomally integrated. At present, BacilloFlex includes a range of multi-functionalB. subtilisspecific parts with applications including metabolic engineering, biosensors and spore surface display. We hope this work will form the foundation of a widely adopted cloning standard forB. subtilisfacilitating collaboration and the sharing of parts.

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Similar and Divergent Effects of ppGpp and DksA Deficiencies on Transcription in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.01410-08 ◽

2009 ◽

Vol 191 (10) ◽

pp. 3226-3236 ◽

Cited By ~ 69

Author(s):

Anna Åberg ◽

Jorge Fernández-Vázquez ◽

Juan David Cabrer-Panes ◽

Alex Sánchez ◽

Carlos Balsalobre

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Rna Polymerase ◽

Functional Group ◽

Fine Tuning ◽

Concerted Action ◽

Secondary Channel ◽

E Coli ◽

Two Factors

ABSTRACT The concerted action of ppGpp and DksA in transcription has been widely documented. In disparity with this model, phenotypic studies showed that ppGpp and DksA might also have independent and opposing roles in gene expression in Escherichia coli. In this study we used a transcriptomic approach to compare the global transcriptional patterns of gene expression in strains deficient in ppGpp (ppGpp0) and/or DksA (ΔdksA). Approximately 6 and 7% of all genes were significantly affected by more than twofold in ppGpp- and DksA-deficient strains, respectively, increasing to 13% of all genes in the ppGpp0 ΔdksA strain. Although the data indicate that most of the affected genes were copositively or conegatively regulated by ppGpp and DksA, some genes that were independently and/or differentially regulated by the two factors were found. The large functional group of chemotaxis and flagellum synthesis genes were notably differentially affected, with all genes being upregulated in the DksA-deficient strain but 60% of them being downregulated in the ppGpp-deficient strain. Revealingly, mutations in the antipausing Gre factors suppress the upregulation observed in the DksA-deficient strain, emphasizing the importance of the secondary channel of the RNA polymerase for regulation and fine-tuning of gene expression in E. coli.

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Golden Gate Assembly of Aerobic and Anaerobic Microbial Bioreporters

Applied and Environmental Microbiology ◽

10.1128/aem.01485-21 ◽

2021 ◽

Author(s):

Aaron J. Hinz ◽

Benjamin Stenzler ◽

Alexandre J. Poulain

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Synthetic Biology ◽

Reporter Gene ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Fluorescent Microscopy ◽

Genetic Circuit ◽

Regulatory Sequences ◽

Golden Gate

Microbial bioreporters provide direct insight into cellular processes by producing a quantifiable signal dictated by reporter gene expression. The core of a bioreporter is a genetic circuit in which a reporter gene (or operon) is fused to promoter and regulatory sequences that govern its expression. In this study, we develop a system for constructing novel Escherichia coli bioreporters based on Golden Gate assembly, a synthetic biology approach for the rapid and seamless fusion of DNA fragments. Gene circuits are generated by fusing promoter and reporter sequences encoding yellow fluorescent protein, mCherry, bacterial luciferase, and an anaerobically active flavin-based fluorescent protein. We address a barrier to the implementation of Golden Gate assembly by designing a series of compatible destination vectors that can accommodate the assemblies. We validate the approach by measuring the activity of constitutive bioreporters and mercury and arsenic biosensors in quantitative exposure assays. We also demonstrate anaerobic quantification of mercury and arsenic in biosensors that produce flavin-based fluorescent protein, highlighting the expanding range of redox conditions that can be examined by microbial bioreporters. IMPORTANCE Microbial bioreporters are versatile genetic tools with wide-ranging applications, particularly in the field of environmental toxicology. For example, biosensors that produce a signal output in the presence of a specific analyte offer less costly alternatives to analytical methods for the detection of environmental toxins such as mercury and arsenic. Biosensors of specific toxins can also be used to test hypotheses regarding mechanisms of uptake, toxicity, and biotransformation. In this study, we develop an assembly platform that uses a synthetic biology technique to streamline construction of novel Escherichia coli bioreporters that produce fluorescent or luminescent signals either constitutively or in response to mercury and arsenic exposure. Beyond the synthesis of novel biosensors, our assembly platform can be adapted for numerous applications, including labelling bacteria for fluorescent microscopy, developing gene expression systems, and modifying bacterial genomes.

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Genomewide Stabilization of mRNA during a “Feast-to-Famine” Growth Transition in Escherichia coli

mSphere ◽

10.1128/msphere.00276-20 ◽

2020 ◽

Vol 5 (3) ◽

Author(s):

Manon Morin ◽

Brice Enjalbert ◽

Delphine Ropers ◽

Laurence Girbal ◽

Muriel Cocaign-Bousquet

Keyword(s):

Gene Expression ◽

Transcriptional Regulation ◽

Mrna Stability ◽

Posttranscriptional Regulation ◽

Carbon Starvation ◽

Fine Tuning ◽

Content Type ◽

E Coli ◽

Transcript Stability

ABSTRACT Bacteria have to continuously adjust to nutrient fluctuations from favorable to less-favorable conditions and in response to carbon starvation. The glucose-acetate transition followed by carbon starvation is representative of such carbon fluctuations observed in Escherichia coli in many environments. Regulation of gene expression through fine-tuning of mRNA pools constitutes one of the regulation levels required for such a metabolic adaptation. It results from both mRNA transcription and degradation controls. However, the contribution of transcript stability regulation in gene expression is poorly characterized. Using combined transcriptome and mRNA decay analyses, we investigated (i) how transcript stability changes in E. coli during the glucose-acetate-starvation transition and (ii) if these changes contribute to gene expression changes. Our work highlights that transcript stability increases with carbon depletion. Most of the stabilization occurs at the glucose-acetate transition when glucose is exhausted, and then stabilized mRNAs remain stable during acetate consumption and carbon starvation. Meanwhile, expression of most genes is downregulated and we observed three times less gene expression upregulation. Using control analysis theory on 375 genes, we show that most of gene expression regulation is driven by changes in transcription. Although mRNA stabilization is not the controlling phenomenon, it contributes to the emphasis or attenuation of transcriptional regulation. Moreover, upregulation of 18 genes (33% of our studied upregulated set) is governed mainly by transcript stabilization. Because these genes are associated with responses to nutrient changes and stress, this underscores a potentially important role of posttranscriptional regulation in bacterial responses to nutrient starvation. IMPORTANCE The ability to rapidly respond to changing nutrients is crucial for E. coli to survive in many environments, including the gut. Reorganization of gene expression is the first step used by bacteria to adjust their metabolism accordingly. It involves fine-tuning of both transcription (transcriptional regulation) and mRNA stability (posttranscriptional regulation). While the forms of transcriptional regulation have been extensively studied, the role of mRNA stability during a metabolic switch is poorly understood. Investigating E. coli genomewide transcriptome and mRNA stability during metabolic transitions representative of the carbon source fluctuations in many environments, we have documented the role of mRNA stability in the response to nutrient changes. mRNAs are globally stabilized during carbon depletion. For a few genes, this leads directly to expression upregulation. As these genes are regulators of stress responses and metabolism, our work sheds new light on the likely importance of posttranscriptional regulations in response to environmental stress.

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Dual UTR-A novel 5′ untranslated region design for synthetic biology applications

10.1101/775643 ◽

2019 ◽

Author(s):

Simone Balzer Le ◽

Ingerid Onsager ◽

Jon Andreas Lorentzen ◽

Rahmi Lale

Keyword(s):

Gene Expression ◽

Synthetic Biology ◽

De Novo ◽

Expression Profiles ◽

Regulation Of Gene Expression ◽

Design Concept ◽

Fine Tuning ◽

The Novel ◽

Wide Range ◽

Application Potential

ABSTRACTBacterial 5′ untranslated regions of mRNA (UTR) involve in a complex regulation of gene expression; however, the exact sequence features contributing to gene regulation are not yet fully understood. In this study, we report the design of a novel 5′ UTR, dual UTR, utilising the transcriptional and translational characteristics of 5′ UTRs in a single expression cassette. The dual UTR consists of two 5′ UTRs, each separately leading to either increase in transcription or translation of the reporter, that are separated by a spacer region, enabling de novo translation initiation. We rationally create dual UTRs with a wide range of expression profiles and demonstrate the functionality of the novel design concept in Escherichia coli and in Pseudomonas putida using different promoter systems and coding sequences. Overall, we demonstrate the application potential of dual UTR design concept in various synthetic biology applications ranging from fine-tuning of gene expression to maximisation of protein production.

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