Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae
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{"title"=>"Synthetic transcription amplifier system for orthogonal control of gene expression in saccharomyces cerevisiae", "type"=>"journal", "authors"=>[{"first_name"=>"Anssi", "last_name"=>"Rantasalo", "scopus_author_id"=>"57173239000"}, {"first_name"=>"Elena", "last_name"=>"Czeizler", "scopus_author_id"=>"9038430000"}, {"first_name"=>"Riitta", "last_name"=>"Virtanen", "scopus_author_id"=>"57172109300"}, {"first_name"=>"Juho", "last_name"=>"Rousu", "scopus_author_id"=>"55888435000"}, {"first_name"=>"Harri", "last_name"=>"Lähdesmäki", "scopus_author_id"=>"6507121329"}, {"first_name"=>"Merja", "last_name"=>"Penttilä", "scopus_author_id"=>"7005401826"}, {"first_name"=>"Jussi", "last_name"=>"Jäntti", "scopus_author_id"=>"6701390357"}, {"first_name"=>"Dominik", "last_name"=>"Mojzita", "scopus_author_id"=>"14042540700"}], "year"=>2016, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"26901642", "sgr"=>"84960977369", "doi"=>"10.1371/journal.pone.0148320", "scopus"=>"2-s2.0-84960977369", "pui"=>"608919549", "isbn"=>"1932-6203 (Electronic)\r1932-6203 (Linking)", "issn"=>"19326203"}, "id"=>"1a9e3f95-e710-3423-ae9b-adc30df1e45c", "abstract"=>"This work describes the development and characterization of a modular synthetic expression system that provides a broad range of adjustable and predictable expression levels in S. cerevisiae. The system works as a fixed-gain transcription amplifier, where the input signal is transferred via a synthetic transcription factor (sTF) onto a synthetic promoter, containing a defined core promoter, generating a transcription output signal. The system activation is based on the bacterial LexA-DNA-binding domain, a set of modified, modular LexA-binding sites and a selection of transcription activation domains. We show both experimentally and computationally that the tuning of the system is achieved through the selection of three separate modules, each of which enables an adjustable output signal: 1) the transcription-activation domain of the sTF, 2) the binding-site modules in the output promoter, and 3) the core promoter modules which define the transcription initiation site in the output promoter. The system has a novel bidirectional architecture that enables generation of compact, yet versatile expression modules for multiple genes with highly diversified expression levels ranging from negligible to very strong using one synthetic transcription factor. In contrast to most existing modular gene expression regulation systems, the present system is independent from externally added compounds. Furthermore, the established system was minimally affected by the several tested growth conditions. These features suggest that it can be highly useful in large scale biotechnology applications.", "link"=>"http://www.mendeley.com/research/synthetic-transcription-amplifier-system-orthogonal-control-gene-expression-saccharomyces-cerevisiae", "reader_count"=>58, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>2, "Researcher"=>19, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>16, "Student > Postgraduate"=>1, "Student > Master"=>8, "Other"=>2, "Student > Bachelor"=>5}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>2, "Researcher"=>19, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>16, "Student > Postgraduate"=>1, "Student > Master"=>8, "Other"=>2, "Student > Bachelor"=>5}, "reader_count_by_subject_area"=>{"Engineering"=>1, "Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>23, "Agricultural and Biological Sciences"=>28, "Chemical Engineering"=>2, "Chemistry"=>2, "Computer Science"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Chemistry"=>{"Chemistry"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>28}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>23}, "Unspecified"=>{"Unspecified"=>1}, "Chemical Engineering"=>{"Chemical Engineering"=>2}}, "reader_count_by_country"=>{"United States"=>1, "China"=>1, "Finland"=>1}, "group_count"=>2}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/4408060"], "description"=>"<div><p>This work describes the development and characterization of a modular synthetic expression system that provides a broad range of adjustable and predictable expression levels in <i>S</i>. <i>cerevisiae</i>. The system works as a fixed-gain transcription amplifier, where the input signal is transferred via a synthetic transcription factor (sTF) onto a synthetic promoter, containing a defined core promoter, generating a transcription output signal. The system activation is based on the bacterial LexA-DNA-binding domain, a set of modified, modular LexA-binding sites and a selection of transcription activation domains. We show both experimentally and computationally that the tuning of the system is achieved through the selection of three separate modules, each of which enables an adjustable output signal: 1) the transcription-activation domain of the sTF, 2) the binding-site modules in the output promoter, and 3) the core promoter modules which define the transcription initiation site in the output promoter. The system has a novel bidirectional architecture that enables generation of compact, yet versatile expression modules for multiple genes with highly diversified expression levels ranging from negligible to very strong using one synthetic transcription factor. In contrast to most existing modular gene expression regulation systems, the present system is independent from externally added compounds. Furthermore, the established system was minimally affected by the several tested growth conditions. These features suggest that it can be highly useful in large scale biotechnology applications.</p></div>", "links"=>[], "tags"=>["core promoter modules", "output promoter", "novel bidirectional architecture", "transcription factor", "transcription output signal", "Synthetic Transcription Amplifier System", "scale biotechnology applications", "transcription activation domains", "gene expression regulation systems", "transcription initiation site", "expression levels"], "article_id"=>2731792, "categories"=>["Biochemistry", "Medicine", "Cell Biology", "Genetics", "Molecular Biology", "Science Policy", "Biological Sciences not elsewhere classified", "Developmental Biology", "Infectious Diseases", "Computational Biology"], "users"=>["Anssi Rantasalo", "Elena Czeizler", "Riitta Virtanen", "Juho Rousu", "Harri Lähdesmäki", "Merja Penttilä", "Jussi Jäntti", "Dominik Mojzita"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0148320", "stats"=>{"downloads"=>25, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Synthetic_Transcription_Amplifier_System_for_Orthogonal_Control_of_Gene_Expression_in_i_Saccharomyces_cerevisiae_i_/2731792", "title"=>"Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in <i>Saccharomyces cerevisiae</i>", "pos_in_sequence"=>0, "defined_type"=>6, "published_date"=>"2016-02-22 09:26:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/4408153"], "description"=>"<p><b>A)</b> Schemes of the DNA constructs used in B–E. <b>B)</b> Normalised GFP fluorescence of strains with the weak sTF42 and the bidirectional reporter constructs in presence of variable concentrations of methionine. <b>C)</b> The normalised mCherry fluorescence of the strains and conditions shown in B. <b>D)</b> The normalised GFP fluorescence of the strains with the strong sTF16 and the bidirectional reporter constructs in presence of variable concentrations of methionine. The values in absence of methionine are not shown due to apparent toxicity of the highly expressed sTF16 in this condition resulting in poor growth of the strains and inconsistent fluorescent values. <b>E)</b> The normalised mCherry fluorescence of the strains and conditions shown in D. The values represent the averages from at least 3 independent experiments; the standard deviations are not shown for clarity of presentation.</p>", "links"=>[], "tags"=>["core promoter modules", "output promoter", "novel bidirectional architecture", "transcription factor", "transcription output signal", "Synthetic Transcription Amplifier System", "scale biotechnology applications", "transcription activation domains", "gene expression regulation systems", "transcription initiation site", "expression levels"], "article_id"=>2731888, "categories"=>["Biochemistry", "Medicine", "Cell Biology", "Genetics", "Molecular Biology", "Science Policy", "Biological Sciences not elsewhere classified", "Developmental Biology", "Infectious Diseases", "Computational Biology"], "users"=>["Anssi Rantasalo", "Elena Czeizler", "Riitta Virtanen", "Juho Rousu", "Harri Lähdesmäki", "Merja Penttilä", "Jussi Jäntti", "Dominik Mojzita"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0148320.g005", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/The_performance_of_the_inducible_synthetic_transcription_factors_/2731888", "title"=>"The performance of the inducible synthetic transcription factors.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-02-22 09:26:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/4408078"], "description"=>"<p><b>A)</b> The input signal is conveyed to the system in form of an activity of the promoter controlling sTF expression (input pr). This could be a constitutive signal or a signal generated in response to physiological changes in cells, such as metabolic or stress responses, or it can be a signal provided by an engineered synthetic control circuit. The input signal is transformed in a cascade of events including production of the sTF, binding the sTF to its specific sites in the output promoter, assembly of the transcription machinery on the core part of the output promoter (cp), and finally initiation of transcription of target gene. The expression of the target gene is the output signal of the system. <b>B)</b> The components, or modules, of the system significantly contribute to the efficiency of the input signal transformation. The amplification of the input signal is achieved by selecting the modules with specific activities providing predictable output signal which is not influenced by the context of the host genetic regulations (the orthogonal system).</p>", "links"=>[], "tags"=>["core promoter modules", "output promoter", "novel bidirectional architecture", "transcription factor", "transcription output signal", "Synthetic Transcription Amplifier System", "scale biotechnology applications", "transcription activation domains", "gene expression regulation systems", "transcription initiation site", "expression levels"], "article_id"=>2731810, "categories"=>["Biochemistry", "Medicine", "Cell Biology", "Genetics", "Molecular Biology", "Science Policy", "Biological Sciences not elsewhere classified", "Developmental Biology", "Infectious Diseases", "Computational Biology"], "users"=>["Anssi Rantasalo", "Elena Czeizler", "Riitta Virtanen", "Juho Rousu", "Harri Lähdesmäki", "Merja Penttilä", "Jussi Jäntti", "Dominik Mojzita"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0148320.g001", "stats"=>{"downloads"=>2, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/The_expression_amplifier_concept_/2731810", "title"=>"The expression amplifier concept.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-02-22 09:26:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/4408183"], "description"=>"<p><b>A</b>-<b>C)</b> Model predictions for the systems with constitutive sTFs. The red bars correspond to the values predicted by the model while the experimetal values are illustrated by the dots ±standard deviation. In all 3 cases the models corresponding to the systems with 1, 2, and 6 BS were used for parameter estimation while all the other systems (0, 3, 4, and 8 BS) were used for model validation. For the 2 constitutive models with the pBID2-EP (<i>PGK1</i>cp for mCherry) (<b>A</b> and <b>B</b>) we estimated only the kinetic parameter associated to the sTF transcription; all the other parameter values were taken from the model associated to the methionine-induced systems (D and E). For the model associated to the strong constitutive sTF16 system using the pBID2-ED (<i>DAN1</i>cp for mCherry) (<b>C</b>) we only needed to estimate the kinetic values associated to the recruitment of polymerase associated with sTF to the core promoter; all the other values were taken from the previous model. <b>D</b>-<b>E</b>) Model predictions for the systems with inducible sTFs. The red surface corresponds to the values predicted by the model while the experimetal values are illustrated by the dashed line.</p>", "links"=>[], "tags"=>["core promoter modules", "output promoter", "novel bidirectional architecture", "transcription factor", "transcription output signal", "Synthetic Transcription Amplifier System", "scale biotechnology applications", "transcription activation domains", "gene expression regulation systems", "transcription initiation site", "expression levels"], "article_id"=>2731915, "categories"=>["Biochemistry", "Medicine", "Cell Biology", "Genetics", "Molecular Biology", "Science Policy", "Biological Sciences not elsewhere classified", "Developmental Biology", "Infectious Diseases", "Computational Biology"], "users"=>["Anssi Rantasalo", "Elena Czeizler", "Riitta Virtanen", "Juho Rousu", "Harri Lähdesmäki", "Merja Penttilä", "Jussi Jäntti", "Dominik Mojzita"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0148320.g006", "stats"=>{"downloads"=>1, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_i_In_silico_i_modeling_of_the_mCherry_fluorescence_in_selected_systems_/2731915", "title"=>"<i>In silico</i> modeling of the mCherry fluorescence in selected systems.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-02-22 09:26:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/4408126"], "description"=>"<p><b>A)</b> Schemes of the DNA constructs used in B–E. The bidirectional promoters are composed of the outwards oriented core promoters of the genes indicated (for the sequence details see Figure B in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148320#pone.0148320.s001\" target=\"_blank\">S1 File</a>) and the varying number of the LexA-binding sites. <b>B)</b> The normalised fluorescence of the strains with the weak sTF42 and <i>ENO1</i>cp-GFP / <i>PGK1</i>cp-mCherry reporter constructs. <b>C)</b> The normalised fluorescence of the strains with the strong sTF16 and <i>ENO1</i>cp-GFP / <i>PGK1</i>cp-mCherry reporter constructs. <b>D)</b> The normalised fluorescence of the strains with the sTF16 and <i>ENO1</i>cp-GFP / <i>DAN1</i>cp-mCherry reporter constructs. <b>E)</b> The normalised fluorescence of the strains with the sTF16 and <i>ENO1</i>cp-GFP / <i>YLR156W</i>cp-mCherry reporter constructs. All the graphs are showing the same range of values on axes depicting the GFP and mCherry fluorescence as in Figs <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148320#pone.0148320.g002\" target=\"_blank\">2</a> and <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148320#pone.0148320.g006\" target=\"_blank\">6</a>, with exception of the mCherry fluorescence in E, where the range of the axis (in red) was adjusted to visualize negligible fluoresce in these strains. The values represent the averages and the error bars the standard deviations from at least 3 independent experiments.</p>", "links"=>[], "tags"=>["core promoter modules", "output promoter", "novel bidirectional architecture", "transcription factor", "transcription output signal", "Synthetic Transcription Amplifier System", "scale biotechnology applications", "transcription activation domains", "gene expression regulation systems", "transcription initiation site", "expression levels"], "article_id"=>2731858, "categories"=>["Biochemistry", "Medicine", "Cell Biology", "Genetics", "Molecular Biology", "Science Policy", "Biological Sciences not elsewhere classified", "Developmental Biology", "Infectious Diseases", "Computational Biology"], "users"=>["Anssi Rantasalo", "Elena Czeizler", "Riitta Virtanen", "Juho Rousu", "Harri Lähdesmäki", "Merja Penttilä", "Jussi Jäntti", "Dominik Mojzita"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0148320.g003", "stats"=>{"downloads"=>2, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Development_of_the_constitutive_system_/2731858", "title"=>"Development of the constitutive system.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-02-22 09:26:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/4408141"], "description"=>"<p><b>A)</b> The levels of GFP and mCherry mRNAs corresponding to the strains shown in Fig 4C; the sTF16 and <i>ENO1</i>cp-GFP / <i>PGK1</i>cp-mCherry <b>B)</b> The levels of GFP and mCherry mRNAs corresponding to the strains shown in the Fig 4D; the sTF16 and <i>ENO1</i>cp-GFP / <i>DAN1</i>cp-mCherry. <b>C)</b> The levels of GFP and mCherry mRNAs corresponding to the strains shown in the Fig 4E; the sTF16 and <i>ENO1</i>cp-GFP / <i>YLR156W</i>cp-mCherry. The values represent fold-difference relative to the <i>IPP1</i> mRNA as determined by RT-PCR. All the graphs are adjusted to show the same range of values on x-axes depicting the level of transcription, with the exception of mCherry RNA levels in C, where the range of the axis (in red) was adjusted to visualize negligible mCherry expression in these strains. The values represent the averages and the error bars the standard deviations from at least 4 experimental replicates.</p>", "links"=>[], "tags"=>["core promoter modules", "output promoter", "novel bidirectional architecture", "transcription factor", "transcription output signal", "Synthetic Transcription Amplifier System", "scale biotechnology applications", "transcription activation domains", "gene expression regulation systems", "transcription initiation site", "expression levels"], "article_id"=>2731870, "categories"=>["Biochemistry", "Medicine", "Cell Biology", "Genetics", "Molecular Biology", "Science Policy", "Biological Sciences not elsewhere classified", "Developmental Biology", "Infectious Diseases", "Computational Biology"], "users"=>["Anssi Rantasalo", "Elena Czeizler", "Riitta Virtanen", "Juho Rousu", "Harri Lähdesmäki", "Merja Penttilä", "Jussi Jäntti", "Dominik Mojzita"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0148320.g004", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Transcription_analysis_of_different_synthetic_promoters_/2731870", "title"=>"Transcription analysis of different synthetic promoters.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-02-22 09:26:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/4408105"], "description"=>"<p><b>A)</b> Schemes of the DNA constructs used in B and C. The bidirectional promoters are composed of the outward oriented core promoters of the indicated genes (for the sequence details see Figure B in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148320#pone.0148320.s001\" target=\"_blank\">S1 File</a>) and six LexA-binding sites inserted between the core promoters. <b>B)</b> The normalised fluorescence signal of the strains grown in presence of 2% D-glucose and in absence of methionine. The fluorescence signal is shown for each core promoter individually. <b>C)</b> Assessment of the background activities of core promoters. The control strains with the same set of the genome-integrated pBID1 reporter expression cassettes as in B, but without the sTF expression cassette. <b>D)</b> Schemes of the constructs used in E and F. The reporter cassettes with full-length promoters assembled in bidirectional orientation. <b>E)</b> The normalised fluorescence signal of the strains grown in presence of 2% D-glucose and in absence of methionine. <b>F)</b> The normalised fluorescence of the strains grown in presence of 2% D-galactose and in absence of methionine. The values represent the averages and the error bars the standard deviations from at least 3 independent experiments.</p>", "links"=>[], "tags"=>["core promoter modules", "output promoter", "novel bidirectional architecture", "transcription factor", "transcription output signal", "Synthetic Transcription Amplifier System", "scale biotechnology applications", "transcription activation domains", "gene expression regulation systems", "transcription initiation site", "expression levels"], "article_id"=>2731837, "categories"=>["Biochemistry", "Medicine", "Cell Biology", "Genetics", "Molecular Biology", "Science Policy", "Biological Sciences not elsewhere classified", "Developmental Biology", "Infectious Diseases", "Computational Biology"], "users"=>["Anssi Rantasalo", "Elena Czeizler", "Riitta Virtanen", "Juho Rousu", "Harri Lähdesmäki", "Merja Penttilä", "Jussi Jäntti", "Dominik Mojzita"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0148320.g002", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Characterization_of_the_core_promoters_/2731837", "title"=>"Characterization of the core promoters.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-02-22 09:26:17"}

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{"start_date"=>"2016-01-01T00:00:00Z", "end_date"=>"2016-12-31T00:00:00Z", "subject_areas"=>[]}
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