RESUMO
KEY MESSAGE: Water deficit-inducible synthetic promoters, SD9-2 and SD18-1, designed for use in the dicot poplar, are functional in the monocot crop, rice.
Assuntos
Oryza , Oryza/genética , Secas , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas/genética , Regulação da Expressão Gênica de PlantasRESUMO
Genomic integration is commonly used to engineer stable production hosts. However, so far, for many microbial workhorses, only a few integration sites have been characterized, thereby restraining advanced strain engineering that requires multiple insertions. Here, we report on the identification of novel genomic integration sites, so-called landing pads, for Pseudomonas putida KT2440. We identified genomic regions with constant expression patterns under diverse experimental conditions by using RNA-Seq data. Homologous recombination constructs were designed to insert heterologous genes into intergenic sites in these regions, allowing condition-independent gene expression. Ten potential landing pads were characterized using four different msfGFP expression cassettes. An insulated probe sensor was used to study locus-dependent effects on recombinant gene expression, excluding genomic read-through of flanking promoters under changing cultivation conditions. While the reproducibility of expression in the landing pads was very high, the msfGFP signals varied strongly between the different landing pads, confirming a strong influence of the genomic context. To showcase that the identified landing pads are also suitable candidates for heterologous gene expression in other Pseudomonads, four equivalent landing pads were identified and characterized in Pseudomonas taiwanensis VLB120. This study shows that genomic "hot" and "cold" spots exist, causing strong promoter-independent variations in gene expression. This highlights that the genomic context is an additional parameter to consider when designing integrable genomic cassettes for tailored heterologous expression. The set of characterized genomic landing pads presented here further increases the genetic toolbox for deep metabolic engineering in Pseudomonads.
Assuntos
Pseudomonas putida , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Perfilação da Expressão Gênica/métodos , Regiões Promotoras Genéticas/genética , Genoma Bacteriano/genética , Recombinação Homóloga , Transcriptoma/genéticaRESUMO
Synthetic promoters are powerful tools to boost the biotechnological potential of microalgae as eco-sustainable industrial hosts. The increasing availability of transcriptome data on microalgae in a variety of environmental conditions allows to identify cis-regulatory elements (CREs) that are responsible for the transcriptional output. Furthermore, advanced cloning technologies, such as golden gate-based MoClo toolkits, enable the creation of modular constructs for testing multiple promoters and a range of reporter systems in a convenient manner. In this chapter, we will describe how to introduce in silico-identified CREs into promoter sequences, and how to clone the modified promoters into MoClo compatible vectors. We will then describe how these promoters can be evaluated and screened for transgene expression in an established microalgal model for genetic perturbation, i.e., Chlamydomonas reinhardtii.
Assuntos
Chlamydomonas reinhardtii , Regiões Promotoras Genéticas , Chlamydomonas reinhardtii/genética , Vetores Genéticos/genética , Clonagem Molecular/métodos , Transgenes , Biologia Sintética/métodos , Microalgas/genética , Engenharia Genética/métodosRESUMO
We discuss the formalism of chemical reaction networks (CRNs) as a computer-aided design interface for using formal methods in engineering living technologies. We set out by reviewing formal methods within a broader view of synthetic biology. Based on published results, we illustrate, step by step, how mathematical and computational techniques on CRNs can be used to study the structural and dynamic properties of the designed systems. As a case study, we use an E. coli two-component system that relays the external inorganic phosphate concentration signal to genetic components. We show how CRN models can scan and explore phenotypic regimes of synthetic promoters with varying detection thresholds, thereby providing a means for fine-tuning the promoter strength to match the specification.