Search

A key driver of synthetic biology is the development of designable genetic parts with predictable behaviors that can be quickly implemented in complex genetic systems. However, the intrinsic complexity of gene regulation can make the rational design of genetic parts challenging. This challenge is apparent in the design of antisense RNA (asRNA) regulators. Though asRNAs are well-known regulators, the literature governing their design is conflicting and leaves the synthetic biology community without clear asRNA design rules. The goal of this study is to perform a comprehensive experimental characterization and statistical analysis of 121 unique asRNA regulators in order to resolve the conflicts that currently exist in the literature. asRNAs usually consist of two regions, the Hfq binding site and the target binding region (TBR). First, the behaviors of several high-performing Hfq binding sites were compared, in terms of their ability to improve repression efficiencies and their orthogonality. Next, a large-scale analysis of TBR design parameters identified asRNA length, the thermodynamics of asRNA-mRNA complex formation, and the percent of target mismatch as key parameters for TBR design. These parameters were used to develop simple asRNA design rules. Finally, these design rules were applied to construct both a simple and a complex genetic circuit containing different asRNAs, and predictable behavior was observed in both circuits. The results presented in this study will drive synthetic biology forward by providing useful design guidelines for the construction of asRNA regulators with predictable behaviors.

Synthetic biology has the potential to provide decisive advances in genetic control of metabolic pathways. However, there are several challenges that synthetic biologists must overcome before this vision becomes a reality. First, a library of diverse and well-characterized sensors, such as metabolite-sensing or condition-sensing promoters, must be constructed. Second, robust programmable circuits that link input conditions with a specific gene regulation response must be developed. Finally, multi-gene targeting strategies must be integrated with metabolically relevant sensors and complex, robust logic. Achievements in each of these areas, which employ the CRISPR/Cas system, in silico modeling, and dynamic sensor-regulators, among other tools, provide a strong basis for future research. Overall, the future for synthetic biology approaches in metabolic engineering holds immense promise.

RNA-based temperature sensing is common in bacteria that live in fluctuating environments. Most naturally-occurring RNA thermosensors are heat-inducible, have long sequences, and function by sequestering the ribosome binding site in a hairpin structure at lower temperatures. Here, we demonstrate the de novo design of short, heat-repressible RNA thermosensors. These thermosensors contain a cleavage site for RNase E, an enzyme native to Escherichia coli and many other organisms, in the 5′ untranslated region of the target gene. At low temperatures, the cleavage site is sequestered in a stem–loop, and gene expression is unobstructed. At high temperatures, the stem–loop unfolds, allowing for mRNA degradation and turning off expression. We demonstrated that these thermosensors respond specifically to temperature and provided experimental support for the central role of RNase E in the mechanism. We also demonstrated the modularity of these RNA thermosensors by constructing a three-input composite circuit that utilizes transcriptional, post-transcriptional, and post-translational regulation. A thorough analysis of the 24 thermosensors allowed for the development of design guidelines for systematic construction of similar thermosensors in future applications. These short, modular RNA thermosensors can be applied to the construction of complex genetic circuits, facilitating rational reprogramming of cellular processes for synthetic biology applications.

Synechocystis sp. strain PCC 6803 is the most widely studied model cyanobacterium, with a well-developed omics level knowledgebase. Like the lifestyles of other cyanobacteria, that of Synechocystis PCC 6803 is tuned to diurnal changes in light intensity. In this study, we analyzed the expression patterns of all of the genes of this cyanobacterium over two consecutive diurnal periods. Using stringent criteria, we determined that the transcript levels of nearly 40% of the genes in Synechocystis PCC 6803 show robust diurnal oscillating behavior, with a majority of the transcripts being upregulated during the early light period. Such transcripts corresponded to a wide array of cellular processes, such as light harvesting, photosynthetic light and dark reactions, and central carbon metabolism. In contrast, transcripts of membrane transporters for transition metals involved in the photosynthetic electron transport chain (e.g., iron, manganese, and copper) were significantly upregulated during the late dark period. Thus, the pattern of global gene expression led to the development of two distinct transcriptional networks of coregulated oscillatory genes. These networks help describe how Synechocystis PCC 6803 regulates its metabolism toward the end of the dark period in anticipation of efficient photosynthesis during the early light period. Furthermore, in silico flux prediction of important cellular processes and experimental measurements of cellular ATP, NADP(H), and glycogen levels showed how this diurnal behavior influences its metabolic characteristics. In particular, NADPH/NADP+ showed a strong correlation with the majority of the genes whose expression peaks in the light. We conclude that this ratio is a key endogenous determinant of the diurnal behavior of this cyanobacterium., IMPORTANCE Cyanobacteria are photosynthetic microbes that use energy from sunlight and CO2 as feedstock. Certain cyanobacterial strains are amenable to facile genetic manipulation, thus enabling synthetic biology and metabolic engineering applications. Such strains are being developed as a chassis for the sustainable production of food, feed, and fuel. To this end, a holistic knowledge of cyanobacterial physiology and its correlation with gene expression patterns under the diurnal cycle is warranted. In this report, a genomewide transcriptional analysis of Synechocystis PCC 6803, the most widely studied model cyanobacterium, sheds light on the global coordination of cellular processes during diurnal periods. Furthermore, we found that, in addition to light, the redox level of NADP(H) is an important endogenous regulator of diurnal entrainment of Synechocystis PCC 6803.

Oil palm (Elaeis guineensis) is the most productive oil bearing crop worldwide. It has three fruit forms, namely dura (thick-shelled), pisifera (shell-less) and tenera (thin-shelled), which are controlled by the SHELL gene. The fruit forms exhibit monogenic co-dominant inheritance, where tenera is a hybrid obtained by crossing maternal dura and paternal pisifera palms. Commercial palm oil production is based on planting thin-shelled tenera palms, which typically yield 30% more oil than dura palms, while pisifera palms are female-sterile and have little to no palm oil yield. It is clear that tenera hybrids produce more oil than either parent due to single gene heterosis. The unintentional planting of dura or pisifera palms reduces overall yield and impacts land utilization that would otherwise be devoted to more productive tenera palms. Here, we identify three additional novel mutant alleles of the SHELL gene, which encode a type II MADS-box transcription factor, and determine oil yield via control of shell fruit form phenotype in a manner similar to two previously identified mutant SHELL alleles. Assays encompassing all five mutations account for all dura and pisifera palms analyzed. By assaying for these variants in 10,224 mature palms or seedlings, we report the first large scale accurate genotype-based determination of the fruit forms in independent oil palm planting sites and in the nurseries that supply them throughout Malaysia. The measured non-tenera contamination rate (10.9% overall on a weighted average basis) underscores the importance of SHELL genetic testing of seedlings prior to planting in production fields. By eliminating non-tenera contamination, comprehensive SHELL genetic testing can improve sustainability by increasing yield on existing planted lands. In addition, economic modeling demonstrates that SHELL gene testing will confer substantial annual economic gains to the oil palm industry, to Malaysian gross national income and to Malaysian government tax receipts.

A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA-asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions.

Microorganisms transform inexpensive carbon sources into highly functionalized compounds without toxic by-product generation or significant energy consumption. By redesigning the natural biosynthetic pathways in an industrially suited host, microbial cell factories can produce complex compounds for a variety of industries. Isoprenoids include many medically important compounds such as antioxidants and anticancer and antimalarial drugs, all of which have been produced microbially. While a biosynthetic pathway could be simply transferred to the production host, the titers would become economically feasible when it is rationally designed, built, and optimized through synthetic biology tools. These tools have been implemented by a number of research groups, with new tools pledging further improvements in yields and expansion to new medically relevant compounds. This review focuses on the microbial production of isoprenoids for the health industry and the advancements though synthetic biology.