Design Of Synthetic Promoters Research Articles

Promoter and Terminator Discovery and Engineering.

Control of gene expression is crucial to optimize metabolic pathways and synthetic gene networks. Promoters and terminators are stretches of DNA upstream and downstream (respectively) of genes that control both the rate at which the gene is transcribed and the rate at which mRNA is degraded. As a result, both of these elements control net protein expression from a synthetic construct. Thus, it is highly important to discover and engineer promoters and terminators with desired characteristics. This chapter highlights various approaches taken to catalogue these important synthetic elements. Specifically, early strategies have focused largely on semi-rational techniques such as saturation mutagenesis to diversify native promoters and terminators. Next, in an effort to reduce the length of the synthetic biology design cycle, efforts in the field have turned towards the rational design of synthetic promoters and terminators. In this vein, we cover recently developed methods such as hybrid engineering, high throughput characterization, and thermodynamic modeling which allow finer control in the rational design of novel promoters and terminators. Emphasis is placed on the methodologies used and this chapter showcases the utility of these methods across multiple host organisms.

Functional dissection of a strong and specific microbe-associated molecular pattern-responsive synthetic promoter.

Synthetic promoters are important for temporal and spatial gene expression in transgenic plants. To identify novel microbe-associated molecular pattern (MAMP)-responsive cis-regulatory sequences for synthetic promoter design, a combination of bioinformatics and experimental approaches was employed. One cis-sequence was identified which confers strong MAMP-responsive reporter gene activity with low background activity. The 35-bp-long cis-sequence was identified in the promoter of the Arabidopsis thaliana DJ1E gene, a homologue of the human oncogene DJ1. In this study, this cis-sequence is shown to be a tripartite cis-regulatory module (CRM). A synthetic promoter with four copies of the CRM linked to a minimal promoter increases MAMP-responsive reporter gene expression compared to the wild-type DJ1E promoter. The CRM consists of two WT-boxes (GGACTTTT and GGACTTTG) and a variant of the GCC-box (GCCACC), all required for MAMP and salicylic acid (SA) responsivity. Yeast one-hybrid screenings using a transcription factor (TF)-only prey library identified two AP2/ERFs, ORA59 and ERF10, interacting antagonistically with the CRM. ORA59 activates reporter gene activity and requires the consensus core sequence GCCNCC for gene expression activation. ERF10 down-regulates MAMP-responsive gene expression. No TFs interacting with the WT-boxes GGACTTTT and GGACTTTG were selected in yeast one-hybrid screenings with the TF-only prey library. In transgenic Arabidopsis, the synthetic promoter confers strong and specific reporter gene activity in response to biotrophs and necrotrophs as well as SA.

Computational discovery of soybean promoter cis-regulatory elements for the construction of soybean cyst nematode-inducible synthetic promoters.

Computational methods offer great hope but limited accuracy in the prediction of functional cis-regulatory elements; improvements are needed to enable synthetic promoter design. We applied an ensemble strategy for de novo soybean cyst nematode (SCN)-inducible motif discovery among promoters of 18 co-expressed soybean genes that were selected from six reported microarray studies involving a compatible soybean-SCN interaction. A total of 116 overlapping motif regions (OMRs) were discovered bioinformatically that were identified by at least four out of seven bioinformatic tools. Using synthetic promoters, the inducibility of each OMR or motif itself was evaluated by co-localization of gain of function of an orange fluorescent protein reporter and the presence of SCN in transgenic soybean hairy roots. Among 16 OMRs detected from two experimentally confirmed SCN-inducible promoters, 11 OMRs (i.e. 68.75%) were experimentally confirmed to be SCN-inducible, leading to the discovery of 23 core motifs of 5- to 7-bp length, of which 14 are novel in plants. We found that a combination of the three best tools (i.e. SCOPE, W-AlignACE and Weeder) could detect all 23 core motifs. Thus, this strategy is a high-throughput approach for de novo motif discovery in soybean and offers great potential for novel motif discovery and synthetic promoter engineering for any plant and trait in crop biotechnology.

Design of synthetic yeast promoters via tuning of nucleosome architecture.

Model-based design of biological parts is a critical goal of synthetic biology, especially for eukaryotes. Here we demonstrate that nucleosome architecture can play a role in defining yeast promoter activity and utilize a computationally-guided approach that can enable both the redesign of endogenous promoter sequences and the de novo design of synthetic promoters. Initially, we use our approach to reprogram native promoters for increased expression and evaluate their performance in various genetic contexts. Increases in expression ranging from 1.5 to nearly 6-fold in a plasmid-based system and up to 16-fold in a genomic context were obtained. Next, we demonstrate that, in a single design cycle, it is possible to create functional, purely synthetic yeast promoters that achieve substantial expression levels (within the top sixth percentile among native yeast promoters). In doing so, this work establishes a unique DNA-level specification of promoter activity and demonstrates predictive design of synthetic parts.

Database Construction for PromoterCAD: Synthetic Promoter Design for Mammals and Plants

Synthetic promoters can control a gene's timing, location, and expression level. The PromoterCAD web server ( http://promotercad.org ) allows the design of synthetic promoters to control plant gene expression, by novel arrangement of cis-regulatory elements. Recently, we have expanded PromoterCAD's scope with additional plant and animal data: (1) PLACE (Plant Cis-acting Regulatory DNA Elements), including various sized sequence motifs; (2) PEDB (Mammalian Promoter/Enhancer Database), including gene expression data for mammalian tissues. The plant PromoterCAD data now contains 22 000 Arabidopsis thaliana genes, 2 200 000 microarray measurements in 20 growth conditions and 79 tissue organs and developmental stages, while the new mammalian PromoterCAD data contains 679 Mus musculus genes and 65 000 microarray measurements in 96 tissue organs and cell types ( http://promotercad.org/mammal/ ). This work presents step-by-step instructions for adding both regulatory motif and gene expression data to PromoterCAD, to illustrate how users can expand PromoterCAD functionality for their own applications and organisms.

508. Synthetic Promoter Design of Cell Type-Specific Adeno-Associated Viral Vectors in the Retina

508. Synthetic Promoter Design of Cell Type-Specific Adeno-Associated Viral Vectors in the Retina

Perspectives on Synthetic Promoters for Biocatalysis and Biotransformation

Acting on the transcriptional level, synthetic promoters have been useful tools for controlling gene expression and have applications in many fields. Here, we discuss synthetic promoters and libraries in regard to current and future applications in the field of biocatalysis or biotransformation. We also focus on synthetic promoter design principles and distinguish between prokaryotic and eukaryotic destinations. The natural toolboxes available for tuneable gene expression and the regulation of enzyme function are limited and primarily host specific. Synthetic biology offers generally applicable concepts and quick implementation. Smart alternatives to transcriptional regulation enrich the engineer's tool box for optimizing industrial enzyme production and host-cell physiology for whole-cell processes. Industrially applicable, tuneable enzyme cascades and artificial circuits for iterative up- and down-regulation will soon be achieved.

Chapter 7: General legal and regulatory issues

This chapter is focused on the methods to design and characterize synthetic promoters derived from and applied in different organisms. We describe general principles of promoter organization, engineering, and characterization based on the reductionist understanding of promoter functions and minimal promoter features. In addition, the particular features of the promoters of commonly used models and other organisms from different life domains are analyzed, as well as the methods for characterization utilized in more notable cases. Methods for the design of synthetic promoters and their application in different scenarios are discussed in the context of actually developed working genetic parts. Finally, the chapter concludes with considerations regarding the possible future developments in the field.