Composability of regulatory sequences controlling transcription and translation in Escherichia coli

Abstract

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...

Variant Interpretation: Functional Assays to the Rescue

“…14,47,48 MAVEs have also been developed to probe the effects of variants in untranslated regions of mRNAs on message stability and protein expression. [16][17][18] Variation in transcriptional regulatory elements is also important, and pathogenic regulatory variants have been identified for a number of Mendelian disorders. [49][50][51][52] One specific example is the alteration of SORT1 (MIM: 602458) expression by a transcriptional regulatory variant that can increase the risk of myocardial infarction by 40%.…”

“…14,15 The effect of variants on mRNA stability and translation can also be measured by multiplex assays. [16][17][18] Finally, deep mutational scans query the effect of amino acid substitutions on protein function. 19 Because these multiplexed functional assays have the capacity to test 10 4 -10 6 variants per experiment, they are already at the scale required for tackling the VUS problem.…”

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

“…The activity of a promoter depends on the sequences that surround it, in particular, the sequences upstream of the -35 site and downstream of the -10 site a↵ect transcription initiation and promoter escape, respectively [14]. The strength of a ribosome binding site is a↵ected by interactions with the 5' UTR sequence and by complicated secondary structures that form across the 5' UTR sequence and the initial gene coding sequence [15] ( Figure 1A). These facts confound system design because, for example, the same combination of promoter and ribosome binding site will result in di↵erent and unpredictable protein production rates depending on the specific gene being expressed.…”

Biología sintética: la ingeniería al asalto de la complejidad biológica

“…Pero para hacer una casa los troncos deben convertirse en vigas y paneles de dimensiones precisas que permitan la construcción a149 de un edificio más complejo. Mediante este razonamiento, una de las señas de identidad de la Biología sintética es el esfuerzo por partir de secuencias de ADN que determinan funciones deseables y modificarlas para que puedan utilizarse como bloques de construcción (bio-ladrillos) de nuevos objetos bioló-gicos (Kosuri et al, 2013;Mutalik et al, 2013). Partiendo de la situación existente, uno puede pensar en modularizar más y más las funciones y los componentes biológicos para hacerlos más fáciles de combinar, tanto física como funcionalmente.…”