Vivek K. Mutalik

Precise and reliable gene expression via standard transcription and translation initiation elements

An inability to reliably predict quantitative behaviors for novel combinations of genetic elements limits the rational engineering of biological systems. We developed an expression cassette architecture for genetic elements controlling transcription and translation initiation in Escherichia coli: transcription elements encode a common mRNA start, and translation elements use an overlapping genetic motif found in many natural systems. We engineered libraries of constitutive and repressor-regulated promoters along with translation initiation elements following these definitions. We measured activity distributions for each library and selected elements that collectively resulted in expression across a 1,000-fold observed dynamic range. We studied all combinations of curated elements, demonstrating that arbitrary genes are reliably expressed to within twofold relative target expression windows with ∼93% reliability. We expect the genetic element definitions validated here can be collectively expanded to create collections of public-domain standard biological parts that support reliable forward engineering of gene expression at genome scales.

Versatile RNA-sensing transcriptional regulators for engineering genetic networks

The widespread natural ability of RNA to sense small molecules and regulate genes has become an important tool for synthetic biology in applications as diverse as environmental sensing and metabolic engineering. Previous work in RNA synthetic biology has engineered RNA mechanisms that independently regulate multiple targets and integrate regulatory signals. However, intracellular regulatory networks built with these systems have required proteins to propagate regulatory signals. In this work, we remove this requirement and expand the RNA synthetic biology toolkit by engineering three unique features of the plasmid pT181 antisense-RNA-mediated transcription attenuation mechanism. First, because the antisense RNA mechanism relies on RNA-RNA interactions, we show how the specificity of the natural system can be engineered to create variants that independently regulate multiple targets in the same cell. Second, because the pT181 mechanism controls transcription, we show how independently acting variants can be configured in tandem to integrate regulatory signals and perform genetic logic. Finally, because both the input and output of the attenuator is RNA, we show how these variants can be configured to directly propagate RNA regulatory signals by constructing an RNA-meditated transcriptional cascade. The combination of these three features within a single RNA-based regulatory mechanism has the potential to simplify the design and construction of genetic networks by directly propagating signals as RNA molecules.

Composability of regulatory sequences controlling transcription and translation in Escherichia coli

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.

Rationally designed families of orthogonal RNA regulators of translation

Our ability to routinely engineer genetic networks for applications is limited by the scarcity of highly specific and non-cross-reacting (orthogonal) gene regulators with predictable behavior. Though antisense RNAs are attractive contenders for this purpose, quantitative understanding of their specificity and sequence-function relationship sufficient for their design has been limited. Here, we use rationally designed variants of the RNA-IN-RNA-OUT antisense RNA-mediated translation system from the insertion sequence IS10 to quantify >500 RNA-RNA interactions in Escherichia coli and integrate the data set with sequence-activity modeling to identify the thermodynamic stability of the duplex and the seed region as the key determinants of specificity. Applying this model, we predict the performance of an additional ~2,600 antisense-regulator pairs, forecast the possibility of large families of orthogonal mutants, and forward engineer and experimentally validate two RNA pairs orthogonal to an existing group of five from the training data set. We discuss the potential use of these regulators in next-generation synthetic biology applications.

Measurement and modeling of intrinsic transcription terminators

The reliable forward engineering of genetic systems remains limited by the ad hoc reuse of many types of basic genetic elements. Although a few intrinsic prokaryotic transcription terminators are used routinely, termination efficiencies have not been studied systematically. Here, we developed and validated a genetic architecture that enables reliable measurement of termination efficiencies. We then assembled a collection of 61 natural and synthetic terminators that collectively encode termination efficiencies across an ∼800-fold dynamic range within Escherichia coli. We simulated co-transcriptional RNA folding dynamics to identify competing secondary structures that might interfere with terminator folding kinetics or impact termination activity. We found that structures extending beyond the core terminator stem are likely to increase terminator activity. By excluding terminators encoding such context-confounding elements, we were able to develop a linear sequence-function model that can be used to estimate termination efficiencies (r = 0.9, n = 31) better than models trained on all terminators (r = 0.67, n = 54). The resulting systematically measured collection of terminators should improve the engineering of synthetic genetic systems and also advance quantitative modeling of transcription termination.

Engineering naturally occurring trans-acting non-coding RNAs to sense molecular signals

Non-coding RNAs (ncRNAs) are versatile regulators in cellular networks. While most trans-acting ncRNAs possess well-defined mechanisms that can regulate transcription or translation, they generally lack the ability to directly sense cellular signals. In this work, we describe a set of design principles for fusing ncRNAs to RNA aptamers to engineer allosteric RNA fusion molecules that modulate the activity of ncRNAs in a ligand-inducible way in Escherichia coli. We apply these principles to ncRNA regulators that can regulate translation (IS10 ncRNA) and transcription (pT181 ncRNA), and demonstrate that our design strategy exhibits high modularity between the aptamer ligand-sensing motif and the ncRNA target-recognition motif, which allows us to reconfigure these two motifs to engineer orthogonally acting fusion molecules that respond to different ligands and regulate different targets in the same cell. Finally, we show that the same ncRNA fused with different sensing domains results in a sensory-level NOR gate that integrates multiple input signals to perform genetic logic. These ligand-sensing ncRNA regulators provide useful tools to modulate the activity of structurally related families of ncRNAs, and building upon the growing body of RNA synthetic biology, our ability to design aptamer–ncRNA fusion molecules offers new ways to engineer ligand-sensing regulatory circuits.

Predicting strength and function for promoters of the Escherichia coli alternative sigma factor, σE

Sequenced bacterial genomes provide a wealth of information but little understanding of transcriptional regulatory circuits largely because accurate prediction of promoters is difficult. We examined two important issues for accurate promoter prediction: (1) the ability to predict promoter strength and (2) the sequence properties that distinguish between active and weak/inactive promoters. We addressed promoter prediction using natural core promoters recognized by the well-studied alternative sigma factor, Escherichia coli σE, as a representative of group 4 σs, the largest σ group. To evaluate the contribution of sequence to promoter strength and function, we used modular position weight matrix models comprised of each promoter motif and a penalty score for suboptimal motif location. We find that a combination of select modules is moderately predictive of promoter strength and that imposing minimal motif scores distinguished active from weak/inactive promoters. The combined -35/-10 score is the most important predictor of activity. Our models also identified key sequence features associated with active promoters. A conserved “AAC” motif in the -35 region is likely to be a general predictor of function for promoters recognized by group 4 σs. These results provide valuable insights into sequences that govern promoter strength, distinguish active and inactive promoters for the first time, and are applicable to both in vivo and in vitro measures of promoter strength.

An adaptor from translational to transcriptional control enables predictable assembly of complex regulation

Bacterial regulators of transcriptional elongation are versatile units for building custom genetic switches, as they control the expression of both coding and noncoding RNAs, act on multigene operons and can be predictably tethered into higher-order regulatory functions (a property called composability). Yet the less versatile bacterial regulators of translational initiation are substantially easier to engineer. To bypass this tradeoff, we have developed an adaptor that converts regulators of translational initiation into regulators of transcriptional elongation in Escherichia coli. We applied this adaptor to the construction of several transcriptional attenuators and activators, including a small molecule–triggered attenuator and a group of five mutually orthogonal riboregulators that we assembled into NOR gates of two, three or four RNA inputs. Continued application of our adaptor should produce large collections of transcriptional regulators whose inherent composability can facilitate the predictable engineering of complex synthetic circuits.

Tuning Promoter Strengths for Improved Synthesis and Function of Electron Conduits in Escherichia coli

Introduction of the electron transfer complex MtrCAB from Shewanella oneidensis MR-1 into a heterologous host provides a modular and molecularly defined route for electrons to be transferred to an extracellular inorganic solid. However, an Escherichia coli strain expressing this pathway displayed limited control of MtrCAB expression and impaired cell growth. To overcome these limitations and to improve heterologous extracellular electron transfer, we used an E. coli host with a more tunable induction system and a panel of constitutive promoters to generate a library of strains that separately transcribe the mtr and cytochrome c maturation (ccm) operons over 3 orders of magnitude. From this library, we identified strains that show 2.2 times higher levels of MtrC and MtrA and that have improved cell growth. We find that a ~300-fold decrease in the efficiency of MtrC and MtrA synthesis with increasing mtr promoter activity critically limits the maximum expression level of MtrC and MtrA. We also tested the extracellular electron transfer capabilities of a subset of the strains using a three-electrode microbial electrochemical system. Interestingly, the strain with improved cell growth and fewer morphological changes generated the largest maximal current per cfu, rather than the strain with more MtrC and MtrA. This strain also showed ~30-fold greater maximal current per cfu than its ccm-only control strain. Thus, the conditions for optimal MtrCAB expression and anode reduction are distinct, and minimal perturbations to cell morphology are correlated with improved extracellular electron transfer in E. coli.

Toward rational design of bacterial genomes

The advent of genetic engineering-the ability to edit and insert DNA into living organisms-in the latter half of the 20th century created visions of a new era of synthetic biology, where novel biological functions could be designed and implemented for useful purposes. We are witnessing an exciting revolution of scale, wherein technical progresses allow for the manipulation of genetic material at the whole genome level. This will enable the manufacture of increasingly complex genetic designs to solve pressing challenges in health, energy and the environment-if and when such designs can be specified. We argue that the organized development of key common application organisms, engineered for engineerability, and attendant libraries of parts, pathways and standardized manufacturing are necessary for this genome-scale technology to realize its promise.