Quantitative estimation of activity and quality for collections of functional genetic elements

Mutalik, Vivek K.; Guimaraes, Joao C.; Cambray, Guillaume; Anh, Quynh; Christoffersen, Marc Juul; Martin, Lance; Yu, Ayumi; Lam, Colin S.C.; Rodriguez, Cesar; Bennett, Gaymon; Keasling, Jay D.; Endy, Drew; Arkin, Adam P.

doi:10.1038/nmeth.2403

Cited by 196 publications

(199 citation statements)

References 64 publications

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“…We conducted a more detailed ANOVA (27), where both RNA and protein levels are independently determined by both promoter and RBS identity. This model is able to take into account effects such as the dependency of RNA levels on the translation rate.…”

Section: Resultsmentioning

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).…”

mentioning

confidence: 99%

“…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).…”

mentioning

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.

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411

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: Resultsmentioning

confidence: 99%

mentioning

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.

Self Cite

411

453

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“…Although far from being a biological equivalent of the semi-conductor fab, this was the aspiration. Work at the BIOFAB also included research to produce metrological tools (Mutalik et al, 2013a;Mutalik et al, 2013b)-infrastructure for measurement of parts' behaviour.…”

Section: The United Statesmentioning

confidence: 99%

Intentions, Expectations and Institutions: Engineering the Future of Synthetic Biology in the USA and the UK

Schyfter

Calvert

2015

Science as Culture

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Synthetic biology is a field in-the-making: a loosely-defined amalgamation of diverse disciplines, institutions, and practices. Where some practitioners identify as scientists, others consider themselves engineers; while some extol the simplicity of standardised biology, others dismiss it as counterproductive. Three different communities in synthetic biology (epistemics, sceptical constructors and committed engineers) can be distinguished by way of their intentions, practices and promises.Synthetic biologists' promises shape policy-makers' expectations, which in turn shape institutional arrangements. These institutional arrangements then influence practitioners' promises in an iterative fashion. In both the US and the UK, 'committed engineers' have succeeded in gaining support for an engineering-based and industry-centred vision of synthetic biology, which promises applications and economic growth. This group's intentions and promises have influenced policy-makers' expectations, which, in turn, have driven the major institutional developments in synthetic biology in the two countries.However, while the promises of the economic potential of this vision of the field have been embraced at policy levels, other aspects of this vision, such as the importance of enabling infrastructure, are often overlooked. In a sense, committed engineers' promises and rhetoric have been too successful, because they have overshadowed the institutional and infrastructural developments needed to make them a reality.

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“…5A). Moreover, a full factorial ANOVA model was applied to quantify the contribution of the interfered promoter::RBS (interaction term), the promoter, and the RBS to the GFP expression (40). ANOVA revealed that the contribution of promoter::RBS interaction without RiboJ (31%) (Fig.…”

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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Quantitative estimation of activity and quality for collections of functional genetic elements

Cited by 196 publications

References 64 publications

Composability of regulatory sequences controlling transcription and translation in Escherichia coli

Composability of regulatory sequences controlling transcription and translation in Escherichia coli

Intentions, Expectations and Institutions: Engineering the Future of Synthetic Biology in the USA and the UK

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

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