RNA processing enables predictable programming of gene expression

Qi, Lei S.; Haurwitz, R.E.; Shao, Wenjun; Doudna, Jennifer A.; Arkin, Adam P.

doi:10.1038/nbt.2355

Cited by 191 publications

(183 citation statements)

References 34 publications

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“…Highly expressed constructs might impair the growth rate and decrease steady-state dilution of cellular contents, which would lead to an overestimation of transcription and translation strengths. We analyze only promoter and RBS pairings here, but future studies can test large numbers of any composable genetic designs to assess their effectiveness on a broad basis (26,28).…”

Section: Discussionmentioning

confidence: 99%

“…Current efforts have focused on approaches to limit the number of time-consuming steps required to characterize potential interactions and on identifying existing or engineered elements that act predictably when used in combination (26)(27)(28). However, these studies still suggest there are enough idiosyncratic interactions and context effects that it will be necessary to construct and measure many variants of a circuit to achieve desired function (29).…”

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confidence: 99%

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Composability of regulatory sequences controlling transcription and translation in Escherichia coli

Kosuri

Goodman

Cambray

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

410

453

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The inability to predict heterologous gene expression levels precisely hinders our ability to engineer biological systems. Using well-characterized regulatory elements offers a potential solution only if such elements behave predictably when combined. We synthesized 12,563 combinations of common promoters and ribosome binding sites and simultaneously measured DNA, RNA, and protein levels from the entire library. Using a simple model, we found that RNA and protein expression were within twofold of expected levels 80% and 64% of the time, respectively. The large dataset allowed quantitation of global effects, such as translation rate on mRNA stability and mRNA secondary structure on translation rate. However, the worst 5% of constructs deviated from prediction by 13-fold on average, which could hinder large-scale genetic engineering projects. The ease and scale this of approach indicates that rather than relying on prediction or standardization, we can screen synthetic libraries for desired behavior.next-generation sequencing | synthetic biology | systems biology O rganisms can be engineered to produce chemical, material, fuel, and medical products that are often superior to nonbiological alternatives (1). Biotechnologists have sought to discover, improve, and industrialize such products through the use of recombinant DNA technologies (2, 3). In recent years, these efforts have increased in complexity from expressing a few genes at once to optimizing multicomponent circuits and pathways (4-7). To attain desired systems-level function reliably, careful and time-consuming optimization of individual components is required (8-11).To mitigate this slow trial-and-error optimization, two dominant approaches have taken hold. The first approach seeks to predict expression levels by elucidating the biophysical relationships between sequence and function. For example, several groups have modified promoters (12, 13) and ribosome binding sites (RBSs) (14-16) to see how small sequence changes affect transcription or translation. Such studies are fundamentally challenging due to the vastness of sequence space. In addition, because these approaches mostly look at either transcription or translation individually, they are rarely able to investigate interactions between these processes.The second approach uses combinations of individually characterized elements to attain desired expression without directly considering their DNA sequences (17-25). Current efforts have focused on approaches to limit the number of time-consuming steps required to characterize potential interactions and on identifying existing or engineered elements that act predictably when used in combination (26-28). However, these studies still suggest there are enough idiosyncratic interactions and context effects that it will be necessary to construct and measure many variants of a circuit to achieve desired function (29). For larger circuits, such approaches are necessarily limited in scope due to the difficulty in measuring large numbers of combinations (26, 27...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Composability of regulatory sequences controlling transcription and translation in Escherichia coli

Kosuri

Goodman

Cambray

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

410

453

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“…However, the two functional elements might interfere with each other by the promoter escaping process (38) or by the formation of some structures inaccessible to ribosomes on the messenger RNA. Different insulators have been reported to eliminate the unanticipated regulatory element interaction in E. coli (33,39). Here, we introduced the well-elucidated RiboJ insulator between promoter and RBS to improve the predictability of the combination.…”

Section: The Riboj Insulator Enables Predictable Combination Of Promomentioning

confidence: 99%

Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces

Bai

Zhang

Zhao

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

168

197

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There is a great demand for precisely quantitating the expression of genes of interest in synthetic and systems biotechnology as new and fascinating insights into the genetics of streptomycetes have come to light. Here, we developed, for the first time to our knowledge, a quantitative method based on flow cytometry and a superfolder green fluorescent protein (sfGFP) at single-cell resolution in Streptomyces. Single cells of filamentous bacteria were obtained by releasing the protoplasts from the mycelium, and the dead cells could be distinguished from the viable ones by propidium iodide (PI) staining. With this sophisticated quantitative method, some 200 native or synthetic promoters and 200 ribosomal binding sites (RBSs) were characterized in a high-throughput format. Furthermore, an insulator (RiboJ) was recruited to eliminate the interference between promoters and RBSs and improve the modularity of regulatory elements. Seven synthetic promoters with gradient strength were successfully applied in a proof-of-principle approach to activate and overproduce the cryptic lycopene in a predictable manner in Streptomyces avermitilis. Our work therefore presents a quantitative strategy and universal synthetic modular regulatory elements, which will facilitate the functional optimization of gene clusters and the drug discovery process in Streptomyces.synthetic biology | natural product | flow cytometry | single-cell resolution | modular regulatory elements S treptomycetes are well known as the most abundant source of bioactive secondary metabolites (1), including medically important antimicrobial agents [e.g., chloramphenicol from Streptomyces venezuelae (2)], agricultural chemicals [e.g., avermectin from Streptomyces avermitilis (3)], and anticancer agents and immunosuppressants [e.g., rapamycin from Streptomyces hygroscopicus (4)]. However, the increasing difficulty of discovering novel drugs via traditional high-throughput screening and the "one strain many compounds" approach is frustrating pharmaceutical productivity (5, 6). Deciphering the genome sequences of Streptomyces surprisingly established the presence of a plethora of gene clusters encoding for yet-unobserved molecules, even in intensively investigated Streptomyces coelicolor A3 (2), revealing a much higher potential of novel bioactive agent production than originally anticipated (7,8). Therefore, the enormous number of natural products that have been obtained likely represent only a tiny portion of the repertoire of bioactive compounds that can possibly be produced. This has brought about extensive research into applied genomics aimed at investigating these new gene clusters, generally referred to as "cryptic," "silent," or "orphan" (9-11). With data on more than 12,000 in-house draft bacterial genomes, the potential for the discovery of a number of novel chemicals encrypted in silent biosynthetic gene clusters has been detected by genome mining.Many new strategies have been documented for "awakening" poorly expressed and/or silent gene clusters in Strept...

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“…This can be obtained by inserting either a rybozyme or a CRISPR target sequence between the 5' UTR sequence and the ribosome binding site [17,18,19]. Testing of constructs with di↵erent UTR sequences demonstrated that insertion of a CRISPR target sequence substantially reduces variability in protein production rate [18]. Similarly, employment of rybozymes in NOT gates showed robustness to the genetic context [17].…”

Section: Modularity Of Basic Partsmentioning

confidence: 99%

“…To decouple the properties of the promoter from those of the ribosome binding site, the 5' UTR sequence can be physically separated from the ribosome binding site to avoid unpredictable interference between these two. This can be obtained by inserting either a rybozyme or a CRISPR target sequence between the 5' UTR sequence and the ribosome binding site [17,18,19]. Testing of constructs with di↵erent UTR sequences demonstrated that insertion of a CRISPR target sequence substantially reduces variability in protein production rate [18].…”

Section: Modularity Of Basic Partsmentioning

confidence: 99%

Modularity, context-dependence, and insulation in engineered biological circuits

Vecchio¹

2015

Trends in Biotechnology

119

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Abstract. The ability of linking systems together such that they behave as predicted once interacting with each other is an essential requirement for the forward engineering of robust synthetic biological circuits. Unfortunately, because of context dependencies, parts and functional modules often behave unpredictably once interacting in the cellular environment. In this paper, we review some recent advances toward establishing a rigorous engineering framework for insulating parts and modules from their context to improve modularity. Overall, a synergy between engineering better parts and higher-level circuit design that overcome the physical limitations of available parts will be important to resolve the problem of context dependence. Modularity and context-dependence in engineered biological systemsSynthetic biology, that is, the use of molecular biology techniques to forward engineer cellular behavior, is a rising branch of biological research [1]. One aim of the field is to gather a better understanding of natural systems by re-wiring their subsystems and exporting them to new settings. The ability of re-designing living systems has especially potential in the biotechnology industry, promising a number of breakthroughs in human health and the environment. Designing living systems does not simply relay on the engineering of parts, such as proteins or genetic sequences. In contrast, it essentially requires the ability of linking parts together to create sophisticated functionalities. Linking parts together to achieve a predictable behavior is 1

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RNA processing enables predictable programming of gene expression

Cited by 191 publications

References 34 publications

Composability of regulatory sequences controlling transcription and translation in Escherichia coli

Composability of regulatory sequences controlling transcription and translation in Escherichia coli

Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces

Modularity, context-dependence, and insulation in engineered biological circuits

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