Further, the throughput of microbial systems allows for combinatorial screening of elements several orders of magnitude greater than what can be accomplished in plants. The part libraries for similar pathway design in microbial systems are much more mature than in plants, as is the standardization of nomenclature across organisms. It is necessary to identify a well-defined part library of regulatory elements that enables tuning of expression to control the optimal stoichiometry of genes necessary to coordinate complex pathways. With regard to precise metabolic engineering, high expression of all genes is rarely desirable. (18,19) While maximization of expression may be ideal for single enzymes, the installation of complex metabolic pathways requires fine coordination both within the pathway and throughout the plant. Many studies have focused on overexpressing transgenes in plant cells for the production of pharmaceutical proteins and other industrially relevant compounds. While the presence of 5′ UTRs is important in the formation of an active translation complex, sequences at 3′ are used for transcription termination and stabilization of the resulting mRNA. RNA-loop structures of UTRs perform many functions including stability of the resulting transcript. (15) Both 5′ UTRs, also known as leaders, and 3′ UTRs, also known as terminators, are important sites of post-transcriptional modification. These cis-regulatory elements are involved in the modulation of gene expression through binding with particular transcription factors. (13,14) In association with the core promoter that is involved in transcription initiation, upstream cis-regulatory elements (enhancers and silencers) are present in different types, numbers, and orientation. Transgene expression can be constitutive (in the entire plant or in specific organs and tissues) or induced by treatment with a cognate molecule acting as a genetic switch. (12) These genetic elements include promoters and untranslated regions (UTRs) at both 5′ and 3′ ends of the coding region. The current plant synthetic biology toolbox comprises endogenous and heterologous genetic elements, as well as synthetic sequences. Through this effort, a well-curated 37-member part library of plant regulatory elements was characterized, providing the necessary data to standardize construct design for precision metabolic engineering in plants.Ī fundamental aspect for genetic engineering is the rational design of transformation vectors to reach the targeted spatio-temporal levels of transgene expression. Analysis of expression levels in plant canopies, individual leaves, and protoplasts were correlated, indicating that any of the methods could be used to evaluate regulatory elements in plants. As anticipated, a dynamic level of expression was achieved from the library, ranging from near undetectable for the weakest cassette to a ∼200-fold increase for the strongest. Constructs were transiently transfected into Nicotiana benthamiana leaves and expression of a fluorescent reporter was measured from plant canopies, leaves, and protoplasts isolated from transfected plants. Here, we use previously described plant regulatory elements to design, build, and test 91 transgene cassettes for relative expression strength. Of particular concern is the absence of combinatorial analysis of regulatory elements, the long design-build-test cycles associated with transgenic plant analysis, and a lack of naming standardization for cloning parts. While the installation of complex genetic circuits in microorganisms is relatively routine, the synthetic biology toolbox is severely limited in plants.
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