Presentation Title

Genome Integration Dependency of Promoter Activity in E. coli

Faculty Mentor

Sriram Kosuri

Start Date

18-11-2017 10:00 AM

End Date

18-11-2017 10:15 AM

Location

9-277

Session

Bio Sciences 4

Type of Presentation

Oral Talk

Subject Area

biological_agricultural_sciences

Abstract

Comprehensive characterization of the location dependency of regulatory sequences can assist in the fine-tuning of transcription regulatory circuits for use in engineering synthetic biological systems. In bacteria, a major determining factor of gene expression is regulation of transcription initiation by promoter sequences. Recent studies in E. coli have highlighted the significance of genome location in regulating promoter activity and associated gene expression. Differential promoter activity is hypothesized to be caused by variations in transcription regulatory elements, such as DNA density, transcription factor accessibility, gene copy number, or the binding of nucleoid-associated proteins. In contributing to the field, we investigate the chromosomal location dependent nature of promoter activity in E. coli through use of a large-scale massively parallel functional assay and site-directed genome integration. Our results show that genomic location contributes to differential gene expression in complex, but not simple promoter sequences. Furthermore, our results support the hypothesis that variations in the spatial distribution of transcription factors plays a major role in differential gene expression. These results support the consideration of location dependent influences on gene expression in future genome integration studies.

Summary of research results to be presented

Here, we characterize the gene location dependent function of promoter regulatory sequences in E. coli through use of a novel high-throughput multiplex reporter assay and landing pad-mediated genome integration. Using a multiplex high-throughput functional assay we show that the location of landing pad integration sites contributes to differential gene expression. The chromosomal location dependency of gene expression is evident in complex, but not simple promoter sequences. Additionally, we address the contribution of gene copy number and transcription factor binding in facilitating the gene location dependency of promoter activity. To investigate if differential promoter activity is dependent on gene copy number, we designed a synthetic simple promoter sequence approximately 200bp in length, consisting of restriction enzyme sites, -35, -10, spacer, UP element, consensus TSS and a randomized null background. We analyzed the activity of this simple promoter at various prespecified locations throughout the E. coli genome. Our results suggest that in the absence of regulatory DNA-binding machinery, the activity of a simple promoter sequence is independent of chromosomal location. The location independency of simple promoter activity is hypothesized to be a consequent of these sequences lacking transcriptional regulatory protein binding motifs. To confirm this hypothesis, we integrated a diverse synthetic library of 17,835 RNA-seq predicted endogenous promoter sequences into three different genome locations and measured their expression. Our results show that a small subset of our diverse library is statistically differentially expressed between these locations; suggesting the contribution of location-mediated variations in transcription factor binding of promoters on gene expression.

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Nov 18th, 10:00 AM Nov 18th, 10:15 AM

Genome Integration Dependency of Promoter Activity in E. coli

9-277

Comprehensive characterization of the location dependency of regulatory sequences can assist in the fine-tuning of transcription regulatory circuits for use in engineering synthetic biological systems. In bacteria, a major determining factor of gene expression is regulation of transcription initiation by promoter sequences. Recent studies in E. coli have highlighted the significance of genome location in regulating promoter activity and associated gene expression. Differential promoter activity is hypothesized to be caused by variations in transcription regulatory elements, such as DNA density, transcription factor accessibility, gene copy number, or the binding of nucleoid-associated proteins. In contributing to the field, we investigate the chromosomal location dependent nature of promoter activity in E. coli through use of a large-scale massively parallel functional assay and site-directed genome integration. Our results show that genomic location contributes to differential gene expression in complex, but not simple promoter sequences. Furthermore, our results support the hypothesis that variations in the spatial distribution of transcription factors plays a major role in differential gene expression. These results support the consideration of location dependent influences on gene expression in future genome integration studies.