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pET vectors allow inducible expression of recombinant proteins in Escherichia coli. In this system, isopropyl β-d-1-thiogalactopyranoside (IPTG) drives lacUV5 promoter to produce T7 RNA polymerase, simultaneously releasing the suppression of T7lac promoter. T7 RNA polymerase then strongly transcribes the target gene. A lac repressor encoded by lacI in the vector represses the promoters. Despite stringent repression and inducible expression achieved with the pET system, unexpected leaky expression can occur without IPTG induction. Here, by evaluating leaky expression in recombinant cells cultured in various Luria-Bertani (LB) media, prepared using yeast extract and peptone from different suppliers, as well as in five commercial premix-LB media, we confirmed the presence of unknown lac inducers in LB. To explore these inducers, we examined E. coli growth in media comprising yeast extract or peptone. At 4% concentration, five commercial yeast extract and six peptone samples individually allowed E. coli growth equivalent to that in LB medium. We determined the luciferase activity of the luxCDABE operon in the pET vector under these conditions. The presence of different concentrations of inducers was detected in both the yeast extract and peptone. Furthermore, we blended yeast extract and peptone with low or high concentrations of lac inducers. The low-expression blend, used as a basal medium before IPTG addition, allowed leak-free, tightly controlled expression. The high-expression blend was used for constitutive high-expression and pET induction with the basal medium, in lieu of IPTG. These blended media can be used for well-controlled inducible and constitutive expression using the pET system., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

In some proteins, a unique class of nonconserved positions is characterized by their ability to generate diverse functional outcomes through single amino acid substitutions. Due to their ability to tune protein function, accurately identifying such "rheostat" positions is crucial for protein design, for understanding the impact of mutations observed in humans, and for predicting the evolution of pathogen drug resistance. However, identifying rheostat positions has been challenging, due-in part-to the absence of a clear structural relationship with binding sites. In this study, experimental data from our previous study of the Escherichia coli lactose repressor protein (LacI) was used to identify rheostat positions for which mutations tune in vivo EC 50 for the allosteric ligand "IPTG." We next used the rheostat assignments to test the hypothesis that rheostat positions have unique dynamic features that will enable their identification. To that end, we integrated all-atom molecular dynamics simulations with perturbation residue response analysis. Results first revealed distinct dynamic behavior in IPTG-bound LacI compared with apo LacI, which was consistent with IPTG's role as an allosteric inducer. Next, we used a variety of dynamic features to build a classification model that discriminates experimentally characterized rheostat positions in LacI from positions with other types of substitution outcomes. In parallel, we built a second classifier model based on the 3D structural "static" network features of LacI. In comparative studies, the dynamic model better identified rheostat positions that were >8 Å from the binding site. In summary, our study provides insights into the dynamic characteristics of rheostat positions and suggests that models built on dynamic features may be useful for predicting the locations of rheostat positions in a wide range of proteins., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. All rights reserved.)

Resource competition is a driver of gut microbiota composition. Bacteria can outcompete metabolically similar rivals through the limitation of shared growth-fuelling nutrients. The mechanisms underlying this remain unclear for bacteria with identical sets of metabolic genes. Here we analysed the lactose utilization operon in the murine commensal Escherichia coli 8178. Using in vitro and in vivo approaches, we showed that translation of the lactose utilization repressor gene lacI from its native non-canonical GTG start codon increases the basal expression of the lactose utilization cluster, enhancing adaptation to lactose consumption. Consequently, a strain carrying the wild type lacI GTG start codon outperformed the lacI ATG start codon mutant in the mouse intestine. This advantage was attenuated upon limiting host lactose intake through diet shift or altering the mutant frequency, emphasizing the context-dependent effect of a single nucleotide change on the bacterial fitness of a common member of the gut microbiota. Coupled with a genomic analysis highlighting the selection of non-ATG start codons in sugar utilization regulator genes across the Enterobacteriaceae family, our data exposed an unsuspected function of non-canonical start codons in metabolic competition., (© 2024. The Author(s).)

Natural prokaryotic gene repression systems often exploit DNA looping to increase the local concentration of gene repressor proteins at a regulated promoter via contributions from repressor proteins bound at distant sites. Using principles from the Escherichia coli lac operon we design analogous repression systems based on target sequence-programmable Transcription Activator-Like Effector dimer (TALED) proteins. Such engineered switches may be valuable for synthetic biology and therapeutic applications. Previous TALEDs with inducible non-covalent dimerization showed detectable, but limited, DNA loop-based repression due to the repressor protein dimerization equilibrium. Here, we show robust DNA loop-dependent bacterial promoter repression by covalent TALEDs and verify that DNA looping dramatically enhances promoter repression in E. coli. We characterize repression using a thermodynamic model that quantitates this favorable contribution of DNA looping. This analysis unequivocally and quantitatively demonstrates that optimized TALED proteins can drive loop-dependent promoter repression in E. coli comparable to the natural LacI repressor system. This work elucidates key design principles that set the stage for wide application of TALED-dependent DNA loop-based repression of target genes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

After an RNA polymerase reaches a terminator, instead of dissociating from the template, it may diffuse along the DNA and recommence RNA synthesis from the previous or a different promoter. Magnetic tweezers were used to monitor such secondary transcription and determine the effects of low forces assisting or opposing translocation, protein roadblocks, and transcription factors. Remarkably, up to 50% of Escherichia coli (E. coli) RNA polymerases diffused along the DNA after termination. Force biased the direction of diffusion (sliding) and the velocity increased rapidly with force up to 0.7 pN and much more slowly thereafter. Sigma factor 70 (σ 70 ) likely remained associated with the DNA promoting sliding and enabling re-initiation from promoters in either orientation. However, deletions of the α-C-terminal domains severely limited the ability of RNAP to turn around between successive rounds of transcription. The addition of elongation factor NusG, which competes with σ 70 for binding to RNAP, limited additional rounds of transcription. Surprisingly, sliding RNA polymerases blocked by a DNA-bound lac repressor could slowly re-initiate transcription and were not affected by NusG, suggesting a σ-independent pathway. Low forces effectively biased promoter selection suggesting a prominent role for topological entanglements that affect RNA polymerase translocation., (© 2024. The Author(s).)

Optogenetics' advancement has made light induction attractive for controlling biological processes due to its advantages of fine-tunability, reversibility, and low toxicity. The lactose operon induction system, commonly used in Escherichia coli, relies on the binding of lactose or isopropyl β-d-1-thiogalactopyranoside (IPTG) to the lactose repressor protein LacI, playing a pivotal role in controlling the lactose operon. Here, we harnessed the light-responsive light-oxygen-voltage 2 (LOV2) domain from Avena sativa phototropin 1 as a tool for light control and engineered LacI into two light-responsive variants, OptoLacIL and OptoLacID. These variants exhibit direct responsiveness to light and darkness, respectively, eliminating the need for IPTG. Building upon OptoLacI, we constructed two light-controlled E. coli gene expression systems, OptoE.coliLight system and OptoE.coliDark system. These systems enable bifunctional gene expression regulation in E. coli through light manipulation and show superior controllability compared to IPTG-induced systems. We applied the OptoE.coliDark system to protein production and metabolic flux control. Protein production levels are comparable to those induced by IPTG. Notably, the titers of dark-induced production of 1,3-propanediol (1,3-PDO) and ergothioneine exceeded 110% and 60% of those induced by IPTG, respectively. The development of OptoLacI will contribute to the advancement of the field of optogenetic protein engineering, holding substantial potential applications across various fields., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A major obstacle to studying DNA replication is that it involves asynchronous and highly delocalized events. A reversible replication barrier overcomes this limitation and allows replication fork movement to be synchronized and localized, facilitating the study of replication fork function and replication coupled repair. Here we provide details on establishing a reversible replication barrier in vitro and using it to monitor different aspects of DNA replication. DNA template containing an array of lac operator (lacO) sequences is first bound to purified lac repressor (LacR). This substrate is then replicated in vitro using a biochemical replication system, which results in replication forks stalled on either side of the LacR array regardless of when or where they arise. Once replication forks are synchronized at the barrier, isopropyl-β-D-thiogalactopyranoside can be added to disrupt LacR binding so that replication forks synchronously resume synthesis. We describe how this approach can be employed to control replication fork elongation, termination, stalling and uncoupling, as well as assays that can be used to monitor these processes. We also explain how this approach can be adapted to control whether replication forks encounter a DNA lesion on the leading or lagging strand template and whether a converging fork is present. The required reagents can be prepared in 1-2 weeks and experiments using this approach are typically performed over 1-3 d. The main requirements for utilizing the LacR replication barrier are basic biochemical expertise and access to an in vitro system to study DNA replication. Investigators should also be trained in working with radioactive materials., (© 2024. Springer Nature Limited.)

Optimal transcriptional regulatory circuits are expected to exhibit stringent control, maintaining silence in the absence of inducers while exhibiting a broad induction dynamic range upon the addition of effectors. In the P lac /LacI pair, the promoter of the lac operon in Escherichia coli is characterized by its leakiness, attributed to the moderate affinity of LacI for its operator target. In response to this limitation, the LacI regulatory protein underwent engineering to enhance its regulatory properties. The M7 mutant, carrying I79T and N246S mutations, resulted in the lac promoter displaying approximately 95% less leaky expression and a broader induction dynamic range compared to the wild-type LacI. An in-depth analysis of each mutation revealed distinct regulatory profiles. In contrast to the wild-type LacI, the M7 mutant exhibited a tighter binding to the operator sequence, as evidenced by surface plasmon resonance studies. Leveraging the capabilities of the M7 mutant, a high-value sugar biosensor was constructed. This biosensor facilitated the selection of mutant galactosidases with approximately a seven-fold improvement in specific activity for transgalactosylation. Consequently, this advancement enabled enhanced biosynthesis of galacto-oligosaccharides (GOS)., (© 2024 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

The functions of many proteins are associated with interconversions among conformational substates. However, these substates can be difficult to measure experimentally, and determining contributions from hydration changes can be especially difficult. Here, we assessed the use of pressure perturbations to sample the substates accessible to the Escherichia coli lactose repressor protein (LacI) in various liganded forms. In the presence of DNA, the regulatory domain of LacI adopts an Open conformation that, in the absence of DNA, changes to a Closed conformation. Increasing the simulation pressure prevented the transition from an Open to a Closed conformation, in a similar manner to the binding of DNA and anti-inducer, ONPF. The results suggest the hydration of specific residues play a significant role in determining the population of different LacI substates and that simulating pressure perturbation could be useful for assessing the role of hydration changes that accompany functionally-relevant amino acid substitutions., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

Biosafety of engineered bacteria as living therapeutics requires a tight regulation to control the specific delivery of protein effectors, maintaining minimum leakiness in the uninduced (OFF) state and efficient expression in the induced (ON) state. Here, we report a three repressors (3R) genetic circuit that tightly regulates the expression of multiple tac promoters (Ptac) integrated in the chromosome of E. coli and drives the expression of a complex type III secretion system injectisome for therapeutic protein delivery. The 3R genetic switch is based on the tetracycline repressor (TetR), the non-inducible lambda repressor cI (ind-) and a mutant lac repressor (LacI W220F ) with higher activity. The 3R switch was optimized with different protein translation and degradation signals that control the levels of LacI W220F . We demonstrate the ability of an optimized switch to fully repress the strong leakiness of the Ptac promoters in the OFF state while triggering their efficient activation in the ON state with anhydrotetracycline (aTc), an inducer suitable for in vivo use. The implementation of the optimized 3R switch in the engineered synthetic injector E. coli (SIEC) strain boosts expression of injectisomes upon aTc induction, while maintaining a silent OFF state that preserves normal growth in the absence of the inducer. Since Ptac is a commonly used promoter, the 3R switch may have multiple applications for tight control of protein expression in E. coli. In addition, the modularity of the 3R switch may enable its tuning for the control of Ptac promoters with different inducers., (© 2023 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

High-resolution NMR spectroscopy enabled us to characterize allosteric transitions between various functional states of the dimeric Escherichia coli Lac repressor. In the absence of ligands, the dimer exists in a dynamic equilibrium between DNA-bound and inducer-bound conformations. Binding of either effector shifts this equilibrium toward either bound state. Analysis of the ternary complex between repressor, operator DNA, and inducer shows how adding the inducer results in allosteric changes that disrupt the interdomain contacts between the inducer binding and DNA binding domains and how this in turn leads to destabilization of the hinge helices and release of the Lac repressor from the operator. Based on our data, the allosteric mechanism of the induction process is in full agreement with the well-known Monod-Wyman-Changeux model., Competing Interests: Competing interests statement:The authors declare no competing interest.

DNA looping has emerged as a central paradigm of transcriptional regulation, as it is shared across many living systems. One core property of DNA looping-based regulation is its ability to greatly enhance repression or activation of genes with only a few copies of transcriptional regulators. However, this property based on a small number of proteins raises the question of the robustness of such a mechanism with respect to the large intracellular perturbations taking place during growth and division of the cell. Here we address the issue of sensitivity to variations of intracellular parameters of gene regulation by DNA looping. We use the lac system as a prototype to experimentally identify the key features of the robustness of DNA looping in growing Escherichia coli cells. Surprisingly, we observe time intervals of tight repression spanning across division events, which can sometimes exceed 10 generations. Remarkably, the distribution of such long time intervals exhibits memoryless statistics that is mostly insensitive to repressor concentration, cell division events, and the number of distinct loops accessible to the system. By contrast, gene regulation becomes highly sensitive to these perturbations when DNA looping is absent. Using stochastic simulations, we propose that the observed robustness to division emerges from the competition between fast, multiple rebinding events of repressors and slow initiation rate of the RNA polymerase. We argue that fast rebinding events are a direct consequence of DNA looping that ensures robust gene repression across a range of intracellular perturbations.

Some proteins, like the lac repressor (LacI), mediate long-range loops that alter DNA topology and create torsional barriers. During transcription, RNA polymerase generates supercoiling that may facilitate passage through such barriers. We monitored E. coli RNA polymerase progress along templates in conditions that prevented, or favored, 400 bp LacI-mediated DNA looping. Tethered particle motion measurements revealed that RNA polymerase paused longer at unlooped LacI obstacles or those barring entry to a loop than those barring exit from the loop. Enhanced dissociation of a LacI roadblock by the positive supercoiling generated ahead of a transcribing RNA polymerase within a torsion-constrained DNA loop may be responsible for this reduction in pause time. In support of this idea, RNA polymerase transcribed 6-fold more slowly through looped DNA and paused at LacI obstacles for 66% less time on positively supercoiled compared to relaxed templates, especially under increased tension (torque). Positive supercoiling propagating ahead of polymerase facilitated elongation along topologically complex, protein-coated templates., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Sequence-specific binding of proteins to DNA is essential for accessing genetic information. We derive a model that predicts an anticorrelation between the macroscopic association and dissociation rates of DNA binding proteins. We tested the model for thousands of different lac operator sequences with a protein binding microarray and by observing kinetics for individual lac repressor molecules in single-molecule experiments. We found that sequence specificity is mainly governed by the efficiency with which the protein recognizes different targets. The variation in probability of recognizing different targets is at least 1.7 times as large as the variation in microscopic dissociation rates. Modulating the rate of binding instead of the rate of dissociation effectively reduces the risk of the protein being retained on nontarget sequences while searching.

Background: The widespread usage of protein expression systems in Escherichia coli (E. coli) is a workhorse of molecular biology research that has practical applications in biotechnology industry, including the production of pharmaceutical drugs. Various factors can strongly affect the successful construction and stable maintenance of clones and the resulting biosynthesis levels. These include an appropriate selection of recombinant hosts, expression systems, regulation of promoters, the repression level at an uninduced state, growth temperature, codon usage, codon context, mRNA secondary structure, translation kinetics, the presence/absence of chaperons and others. However, optimization of the growth medium's composition is often overlooked. We systematically evaluate this factor, which can have a dramatic effect on the expression of recombinant proteins, especially those which are toxic to a recombinant host., Results: Commonly used animal tissue- and plant-based media were evaluated using a series of clones in pET vector, containing expressed Open Reading Frames (ORFs) with a wide spectrum of toxicity to the recombinant E. coli: (i) gfpuv (nontoxic); (ii) tp84_28-which codes for thermophilic endolysin (moderately toxic); and (iii) tthHB27IRM-which codes for thermophilic restriction endonuclease-methyltransferase (REase-MTase)-RM.TthHB27I (very toxic). The use of plant-derived peptones (soy peptone and malt extract) in a culture medium causes the T7-lac expression system to leak. We show that the presence of raffinose and stachyose (galactoside derivatives) in those peptones causes premature and uncontrolled induction of gene expression, which affects the course of the culture, the stability of clones and biosynthesis levels., Conclusions: The use of plant-derived peptones in a culture medium when using T7-lac hybrid promoter expression systems, such as Tabor-Studier, can lead to uncontrolled production of a recombinant protein. These conclusions also extend to other, lac operator-controlled promoters. In the case of proteins which are toxic to a recombinant host, this can result in mutations or deletions in the expression vector and/or cloned gene, the death of the host or highly decreased expression levels. This phenomenon is caused by the content of certain saccharides in plant peptones, some of which (galactosides) may act as T7-lac promoter inducer by interacting with a Lac repressor. Thus, when attempting to overexpress toxic proteins, it is recommended to either not use plant-derived media or to use them with caution and perform a pilot-scale evaluation of the derepression effect on a case-by-case basis., (© 2022. The Author(s).)

The lactose uptake pathway of E. coli is a paradigmatic example of multistability in gene regulatory circuits. In the induced state of the lac pathway, the genes comprising the lac operon are transcribed, leading to the production of proteins that import and metabolize lactose. In the uninduced state, a stable repressor-DNA loop frequently blocks the transcription of the lac genes. Transitions from one phenotypic state to the other are driven by fluctuations, which arise from the random timing of the binding of ligands and proteins. This stochasticity affects transcription and translation, and ultimately molecular copy numbers. Our aim is to understand the transition from the induced to the uninduced state of the lac operon. We use a detailed computational model to show that repressor-operator binding and unbinding, fluctuations in the total number of repressors, and inducer-repressor binding and unbinding all play a role in this transition. Based on the timescales on which these processes operate, we construct a minimal model of the transition to the uninduced state and compare the results with simulations and experimental observations. The induced state turns out to be very stable, with a transition rate to the uninduced state lower than 2×10 -9 per minute. In contrast to the transition to the induced state, the transition to the uninduced state is well described in terms of a 2D diffusive system crossing a barrier, with the diffusion rates emerging from a model of repressor unbinding., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

When amino acids vary during evolution, the outcome can be functionally neutral or biologically-important. We previously found that substituting a subset of nonconserved positions, "rheostat" positions, can have surprising effects on protein function. Since changes at rheostat positions can facilitate functional evolution or cause disease, more examples are needed to understand their unique biophysical characteristics. Here, we explored whether "phylogenetic" patterns of change in multiple sequence alignments (such as positions with subfamily specific conservation) predict the locations of functional rheostat positions. To that end, we experimentally tested eight phylogenetic positions in human liver pyruvate kinase (hLPYK), using 10-15 substitutions per position and biochemical assays that yielded five functional parameters. Five positions were strongly rheostatic and three were non-neutral. To test the corollary that positions with low phylogenetic scores were not rheostat positions, we combined these phylogenetic positions with previously-identified hLPYK rheostat, "toggle" (most substitution abolished function), and "neutral" (all substitutions were like wild-type) positions. Despite representing 428 variants, this set of 33 positions was poorly statistically powered. Thus, we turned to the in vivo phenotypic dataset for E. coli lactose repressor protein (LacI), which comprised 12-13 substitutions at 329 positions and could be used to identify rheostat, toggle, and neutral positions. Combined hLPYK and LacI results show that positions with strong phylogenetic patterns of change are more likely to exhibit rheostat substitution outcomes than neutral or toggle outcomes. Furthermore, phylogenetic patterns were more successful at identifying rheostat positions than were co-evolutionary or eigenvector centrality measures of evolutionary change., (© 2021 The Protein Society.)

Recent progress in DNA synthesis and sequencing technology has enabled systematic studies of protein function at a massive scale. We explore a deep mutational scanning study that measured the transcriptional repression function of 43,669 variants of the Escherichia coli LacI protein. We analyze structural and evolutionary aspects that relate to how the function of this protein is maintained, including an in-depth look at the C-terminal domain. We develop a deep neural network to predict transcriptional repression mediated by the lac repressor of Escherichia coli using experimental measurements of variant function. When measured across 10 separate training and validation splits using 5,009 single mutations of the lac repressor, our best-performing model achieved a median Pearson correlation of 0.79, exceeding any previous model. We demonstrate that deep representation learning approaches, first trained in an unsupervised manner across millions of diverse proteins, can be fine-tuned in a supervised fashion using lac repressor experimental datasets to more effectively predict a variant's effect on repression. These findings suggest a deep representation learning model may improve the prediction of other important properties of proteins., Competing Interests: Competing interest statement: G.M.C. is a cofounder of Nabla Bio, Inc., in which he has related financial interests. A full list of G.M.C.’s technology transfer, advisory roles, and funding sources is available at arep.med.harvard.edu/gmc/tech.html. A.S.G. and K.M.C. declare no competing interest.

DNA looping plays an important role in cells in both regulating and protecting the genome. Often, studies of looping focus on looping by prokaryotic transcription factors like lac repressor or by structural maintenance of chromosomes proteins such as condensin. Here, however, we are interested in a different looping method whereby condensing agents (charge ≥+3) such as protamine proteins neutralize the DNA, causing it to form loops and toroids. We considered two previously proposed mechanisms for DNA looping by protamine. In the first mechanism, protamine stabilizes spontaneous DNA fluctuations, forming randomly distributed loops along the DNA. In the second mechanism, protamine binds and bends the DNA to form a loop, creating a distribution of loops that is biased by protamine binding. To differentiate between these mechanisms, we imaged both spontaneous and protamine-induced loops on short-length (≤1 μm) DNA fragments using atomic force microscopy. We then compared the spatial distribution of the loops to several model distributions. A random looping model, which describes the mechanism of spontaneous DNA folding, fit the distribution of spontaneous loops, but it did not fit the distribution of protamine-induced loops. Specifically, it failed to predict a peak in the spatial distribution of loops at an intermediate location along the DNA. An electrostatic multibinding model, which was created to mimic the bind-and-bend mechanism of protamine, was a better fit of the distribution of protamine-induced loops. In this model, multiple protamines bind to the DNA electrostatically within a particular region along the DNA to coordinate the formation of a loop. We speculate that these findings will impact our understanding of protamine's in vivo role for looping DNA into toroids and the mechanism of DNA condensation by condensing agents more broadly., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Allostery is a fundamental biophysical mechanism that underlies cellular sensing, signaling, and metabolism. Yet a quantitative understanding of allosteric genotype-phenotype relationships remains elusive. Here, we report the large-scale measurement of the genotype-phenotype landscape for an allosteric protein: the lac repressor from Escherichia coli, LacI. Using a method that combines long-read and short-read DNA sequencing, we quantitatively measure the dose-response curves for nearly 10 5 variants of the LacI genetic sensor. The resulting data provide a quantitative map of the effect of amino acid substitutions on LacI allostery and reveal systematic sequence-structure-function relationships. We find that in many cases, allosteric phenotypes can be quantitatively predicted with additive or neural-network models, but unpredictable changes also occur. For example, we were surprised to discover a new band-stop phenotype that challenges conventional models of allostery and that emerges from combinations of nearly silent amino acid substitutions., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Photolabile protecting groups play a significant role in controlling biological functions and cellular processes in living cells and tissues, as light offers high spatiotemporal control, is non-invasive as well as easily tuneable. In the recent past, photo-responsive inducer molecules such as 6-nitropiperonyl-caged IPTG (NP-cIPTG) have been used as optochemical tools for Lac repressor-controlled microbial expression systems. To further expand the applicability of the versatile optochemical on-switch, we have investigated whether the modulation of cIPTG water solubility can improve the light responsiveness of appropriate expression systems in bacteria. To this end, we developed two new cIPTG derivatives with different hydrophobicity and demonstrated both an easy applicability for the light-mediated control of gene expression and a simple transferability of this optochemical toolbox to the biotechnologically relevant bacteria Pseudomonas putida and Bacillus subtilis. Notably, the more water-soluble cIPTG derivative proved to be particularly suitable for light-mediated gene expression in these alternative expression hosts., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

Transcription factor decoy binding sites are short DNA sequences that can titrate a transcription factor away from its natural binding site, therefore regulating gene expression. In this study, we harness synthetic transcription factor decoy systems to regulate gene expression for metabolic pathways in Escherichia coli. We show that transcription factor decoys can effectively regulate expression of native and heterologous genes. Tunability of the decoy can be engineered via changes in copy number or modifications to the DNA decoy site sequence. Using arginine biosynthesis as a showcase, we observed a 16-fold increase in arginine production when we introduced the decoy system to steer metabolic flux towards increased arginine biosynthesis, with negligible growth differences compared to the wild type strain. The decoy-based production strain retains high genetic integrity; in contrast to a gene knock-out approach where mutations were common, we detected no mutations in the production system using the decoy-based strain. We further show that transcription factor decoys are amenable to multiplexed library screening by demonstrating enhanced tolerance to pinene with a combinatorial decoy library. Our study shows that transcription factor decoy binding sites are a powerful and compact tool for metabolic engineering., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Gene regulation by control of transcription initiation is a fundamental property of living cells. Much of our understanding of gene repression originated from studies of the Escherichia coli lac operon switch, in which DNA looping plays an essential role. To validate and generalize principles from lac for practical applications, we previously described artificial DNA looping driven by designed transcription activator-like effector dimer (TALED) proteins. Because TALE monomers bind the idealized symmetrical lac operator sequence in two orientations, our prior studies detected repression due to multiple DNA loops. We now quantitatively characterize gene repression in living E. coli by a collection of individual TALED loops with systematic loop length variation. Fitting of a thermodynamic model allows unequivocal demonstration of looping and comparison of the engineered TALED repression system with the natural lac repressor system., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Inducible promoters are a central regulatory component in synthetic biology, metabolic engineering, and protein production for laboratory and commercial uses. Many of these applications utilize two or more exogenous promoters, imposing a currently unquantifiable metabolic burden on the living system. Here, we engineered a collection of inducible promoters (regulated by LacI-based transcription factors) that maximize the free-state of endogenous RNA polymerase (RNAP). We leveraged this collection of inducible promotors to construct simple two-channel logical controls that enabled us to measure metabolic burden - as it relates to RNAP resource partitioning. The two-channel genetic circuits utilized sets of signal-coupled transcription factors that regulate cognate inducible promoters in a coordinated logical fashion. With this fundamental genetic architecture, we evaluated the performance of each inducible promoter as discrete operations, and as coupled systems to evaluate and quantify the effects of resource partitioning. Obtaining the ability to systematically and accurately measure the apparent RNA-polymerase resource budget will enable researchers to design more robust genetic circuits, with significantly higher fidelity. Moreover, this study presents a workflow that can be used to better understand how living systems adapt RNAP resources, via the complementary pairing of constitutive and regulated promoters that vary in strength., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Many proteins that bind specific DNA sequences search the genome by combining three-dimensional diffusion with one-dimensional sliding on nonspecific DNA 1-5 . Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore the DNA surface during the one-dimensional phase of target search. To track the rotation of sliding LacI molecules on the microsecond timescale, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluctuations in fluorescence signal are accurately described by rotation-coupled sliding, in which LacI traverses about 40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA; this suggests that the sliding protein frequently hops out of the DNA groove, which would result in the frequent bypassing of target sequences. We directly observe such bypassing using single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI hops one or two grooves (10-20 bp) every 200-700 μs. Our data suggest a trade-off between speed and accuracy during sliding: the weak nature of nonspecific protein-DNA interactions underlies operator bypassing, but also speeds up sliding. We anticipate that SMCT-FCS, which monitors rotational diffusion on the microsecond timescale while tracking individual molecules with millisecond resolution, will be applicable to the real-time investigation of many other biological interactions and will effectively extend the accessible time regime for observing these interactions by two orders of magnitude.

Mobile genetic elements, such as plasmids, phages, and transposons, are important sources for evolution of novel functions. In this study, we performed a large-scale screening of metagenomic phage libraries for their ability to suppress temperature-sensitivity in Salmonella enterica serovar Typhimurium strain LT2 mutants to examine how phage DNA could confer evolutionary novelty to bacteria. We identified an insert encoding 23 amino acids from a phage that when fused with a bacterial DNA-binding repressor protein (LacI) resulted in the formation of a chimeric protein that localized to the outer membrane. This relocalization of the chimeric protein resulted in increased membrane vesicle formation and an associated suppression of the temperature sensitivity of the bacterium. Both the host LacI protein and the extracellular 23-amino acid stretch are necessary for the generation of the novel phenotype. Furthermore, mutational analysis of the chimeric protein showed that although the native repressor function of the LacI protein is maintained in this chimeric structure, it is not necessary for the new function. Thus, our study demonstrates how a gene fusion between foreign DNA and bacterial DNA can generate novelty without compromising the native function of a given gene., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The Lac system of genes has been an important model system in understanding gene regulation. When the dimer lac repressor protein binds to the correct DNA sequence, the hinge region of the protein goes through a disorder to order transition. The hinge region is disordered when binding to nonoperator sequences. This region of the protein must pay a conformational entropic penalty to order when it is bound to operator DNA. Structural studies show that this region is flexible. Previous simulations showed that this region is disordered when free in solution without the DNA binding domain present. Our simulations corroborate that this region is extremely flexible in solution, but we find that the presence of the DNA binding domain proximal to the hinge helix and salt make the ordered conformation more favorable even without DNA present., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Herein we report a new type of artificial cells capable of long-term protein expression and regulation. We constructed the artificial cells by grafting anti-His-tag aptamer into the polymer backbone of the hydrogel particles, and then immobilizing the His-tagged proteinaceous factors of the transcription and translation system into the hydrogel particles. Long-term protein expression for at least 16 days was achieved by continuously flowing feeding buffer through the artificial cells. The effect of various metal ions on the protein expression in the artificial cells was investigated. Utilizing the lac operator-repressor system, we could regulate the expression level of eGFP in the artificial cells by controlling the β-D-1-thiogalatopyranoside (IPTG) concentration in the feeding buffer. The artificial cells based on the aptamer grafted hydrogel provide a useful platform for gene circuit engineering, metabolic engineering, drug delivery, and biosensors.

Bifidobacteria resident in the gastrointestinal tract (GIT) are subject to constantly changing environmental conditions, which require rapid adjustments in gene expression. Here, we show that two predicted LacI-type transcription factors (TFs), designated AraQ and MalR1, are involved in regulating the central, carbohydrate-associated metabolic pathway (the so-called phosphoketolase pathway or bifid shunt) of the gut commensal Bifidobacterium breve UCC2003. These TFs appear to not only control transcription of genes involved in the bifid shunt and each other, but also seem to commonly and directly affect transcription of other TF-encoding genes, as well as genes related to uptake and metabolism of various carbohydrates. This complex and interactive network of AraQ/MalR1-mediated gene regulation provides previously unknown insights into the governance of carbon metabolism in bifidobacteria.

Cells execute complex transcriptional programs by deploying distinct protein regulatory assemblies that interact with cis-regulatory elements throughout the genome. Using concepts from DNA nanotechnology, we synthetically recapitulated this feature in in vitro gene networks actuated by T7 RNA polymerase (RNAP). Our approach involves engineering nucleic acid hybridization interactions between a T7 RNAP site-specifically functionalized with single-stranded DNA (ssDNA), templates displaying cis-regulatory ssDNA domains, and auxiliary nucleic acid assemblies acting as artificial transcription factors (TFs). By relying on nucleic acid hybridization, de novo regulatory assemblies can be computationally designed to emulate features of protein-based TFs, such as cooperativity and combinatorial binding, while offering unique advantages such as programmability, chemical stability, and scalability. We illustrate the use of nucleic acid TFs to implement transcriptional logic, cascading, feedback, and multiplexing. This framework will enable rapid prototyping of increasingly complex in vitro genetic devices for applications such as portable diagnostics, bioanalysis, and the design of adaptive materials.

To bridge biological and biochemical disciplines, the relationship between in vitro protein biochemical function and in vivo activity must be established. Such studies can (a) help determine whether properties measured in simple, dilute solutions extrapolate to the complex in vivo conditions and (b) illuminate cryptic biological factors that are new avenues for study. We have explored the in vivo-in vitro relationship for chimeras built from LacI/GalR transcription regulators. In prior studies of individual chimeras, amino acid changes that altered in vitro DNA binding affinity exhibited correlated changes in in vivo transcription repression. However, discrepancies arose when the two datasets were compared to each other: Although their DNA binding domains were identical and their in vitro binding affinities spanned the same range, their in vivo repression ranges differed by >50-fold. Here, we determined that the presence of endogenous ligand for one chimera further exacerbated the offset, but that different abilities to simultaneously bind and "loop" two DNA operators resolves the discrepancy. Indeed, results suggest that the lac operon can be looped by even weakly interacting repressor dimers. For looping-competent repressors, we measured in vitro binding to the secondary operator. Surprisingly, this was largely insensitive to amino acid changes in the repressor protein, which reflects altered specificity; this supports the emerging view that the locations of specificity determining positions can be unique to each protein homolog. In aggregate, this work illustrates how a comparative approach can enrich understanding of the in vivo-in vitro relationship and suggest unexpected avenues for future study., (© 2019 The Protein Society.)

Mutation is a critical mechanism by which evolution explores the functional landscape of proteins. Despite our ability to experimentally inflict mutations at will, it remains difficult to link sequence-level perturbations to systems-level responses. Here, we present a framework centered on measuring changes in the free energy of the system to link individual mutations in an allosteric transcriptional repressor to the parameters which govern its response. We find that the energetic effects of the mutations can be categorized into several classes which have characteristic curves as a function of the inducer concentration. We experimentally test these diagnostic predictions using the well-characterized LacI repressor of Escherichia coli , probing several mutations in the DNA binding and inducer binding domains. We find that the change in gene expression due to a point mutation can be captured by modifying only the model parameters that describe the respective domain of the wild-type protein. These parameters appear to be insulated, with mutations in the DNA binding domain altering only the DNA affinity and those in the inducer binding domain altering only the allosteric parameters. Changing these subsets of parameters tunes the free energy of the system in a way that is concordant with theoretical expectations. Finally, we show that the induction profiles and resulting free energies associated with pairwise double mutants can be predicted with quantitative accuracy given knowledge of the single mutants, providing an avenue for identifying and quantifying epistatic interactions., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Enhancers are ubiquitous and critical gene-regulatory elements. However, quantitative understanding of the role of DNA looping in the regulation of enhancer action and specificity is limited. We used the Escherichia coli NtrC enhancer-σ54 promoter system as an in vivo model, finding that NtrC activation is highly sensitive to the enhancer-promoter (E-P) distance in the 300-6,000 bp range. DNA loops formed by Lac repressor were able to strongly regulate enhancer action either positively or negatively, recapitulating promoter targeting and insulation. A single LacI loop combining targeting and insulation produced a strong shift in specificity for enhancer choice between two σ54 promoters. A combined kinetic-thermodynamic model was used to quantify the effect of DNA-looping interactions on promoter activity and revealed that sensitivity to E-P distance and to control by other loops is itself dependent on enhancer and promoter parameters that may be subject to regulation., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The proper pausing of replication forks at barriers on chromosomes is important for genome integrity. However, the detailed mechanism underlying this process has not been well elucidated. Here, we successfully reconstituted fork-pausing reactions from purified yeast proteins on templates that had binding sites for the LacI, LexA, and/or Fob1 proteins; the forks paused specifically at the protein-bound sites. Moreover, although the replicative helicase Cdc45-Mcm2-7-GINS (CMG) complex alone unwound the protein-bound templates, the unwinding of the LacI-bound site was impeded by the presence of a main leading strand DNA polymerase: polymerase ε (Polε). This suggests that Polε modulates CMG to pause at these sites., (© 2018 Hizume et al.; Published by Cold Spring Harbor Laboratory Press.)

The engineering of microorganisms to monitor environmental chemicals or to produce desirable bioproducts is often reliant on the availability of a suitable biosensor. However, the conversion of a ligand-binding protein into a biosensor has been difficult. Here, we report a general strategy for generating biosensors in Escherichia coli that act by ligand-dependent stabilization of a transcriptional activator and mediate ligand concentration-dependent expression of a reporter gene. We constructed such a biosensor by using the lac repressor, LacI, as the ligand-binding domain and fusing it to the Zif268 DNA-binding domain and RNA polymerase omega subunit transcription-activating domain. Using error-prone PCR mutagenesis of lacI and selection, we identified a biosensor with multiple mutations, only one of which was essential for biosensor behavior. By tuning parameters of the assay, we obtained a response dependent on the ligand isopropyl β-d-1-thiogalactopyranoside (IPTG) of up to a 7-fold increase in the growth rate of E. coli. The single destabilizing mutation combined with a lacI mutation that expands ligand specificity to d-fucose generated a biosensor with improved response both to d-fucose and to IPTG. However, a mutation equivalent to the one that destabilized LacI in either of two structurally similar periplasmic binding proteins did not confer ligand-dependent stabilization. Finally, we demonstrated the generality of this method by using mutagenesis and selection to engineer another ligand-binding domain, MphR, to function as a biosensor. This strategy may allow many natural proteins that recognize and bind to ligands to be converted into biosensors.

The lac operon is a delicate inducible gene expression element in bacteria. To efficiently induce gene expression, a sufficient dosage of an inducer, usually that of 500-1000 µM isopropyl β-D-1-thiogalactopyranoside (IPTG), is required to keep repressor LacI from its binding sites, which is a heavy cost burden in low-value-added products. So we propose a strategy to reduce the required dosage of IPTG by restricting LacI expression. To test this strategy, we employed a reconstructed IPTG inducible expression system based on lac operon, Promoter(lacO)-target gene-PtacL-lacI, where a modified promoter, Ptac, with a random synthetic library (PtacL) to instead of PlacI to optimize LacI expression in Escherichia coli. Finally, the PtacL mutant, PtacL4, which could maintain the same repression effect as the original PlacI while reducing the required dosage of IPTG from 500 to 20 µM, was selected. This method is simple and efficient and can be of a good reference point for attempts to reduce inducer concentration in the IPTG or similar inducible expression systems., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis-based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein's activity. We find that the redesigned LacI retains a robust activity in vitro and in vivo, provided that the third native cysteine at position 281 is retained in LacI. In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic details of LacI target search at the single molecule level., Competing Interests: The authors have declared that no competing interests exist.

In any living cell, genome maintenance is carried out by DNA-binding proteins that recognize specific sequences among a vast amount of DNA. This includes fundamental processes such as DNA replication, DNA repair, and gene expression and regulation. Here, we study the mechanism of DNA target search by a single lac repressor protein (LacI) with ultrafast force-clamp spectroscopy, a sub-millisecond and few base-pair resolution technique based on laser tweezers. We measure 1D-diffusion of proteins on DNA at physiological salt concentrations with 20 bp resolution and find that sliding of LacI along DNA is sequence dependent. We show that only allosterically activated LacI slides along non-specific DNA sequences during target search, whereas the inhibited conformation does not support sliding and weakly interacts with DNA. Moreover, we find that LacI undergoes a load-dependent conformational change when it switches between sliding and strong binding to the target sequence. Our data reveal how DNA sequence and molecular switching regulate LacI target search process and provide a comprehensive model of facilitated diffusion for LacI.

Supercoiling can alter the form and base pairing of the double helix and directly impact protein binding. More indirectly, changes in protein binding and the stress of supercoiling also influence the thermodynamic stability of regulatory, protein-mediated loops and shift the equilibria of fundamental DNA/chromatin transactions. For example, supercoiling affects the hierarchical organization and function of chromatin in topologically associating domains (TADs) in both eukaryotes and bacteria. On the other hand, a protein-mediated loop in DNA can constrain supercoiling within a plectonemic structure. To characterize the extent of constrained supercoiling, 400 bp, lac repressor-secured loops were formed in extensively over- or under-wound DNA under gentle tension in a magnetic tweezer. The protein-mediated loops constrained variable amounts of supercoiling that often exceeded the maximum writhe expected for a 400 bp plectoneme. Loops with such high levels of supercoiling appear to be entangled with flanking domains. Thus, loop-mediating proteins operating on supercoiled substrates can establish topological domains that may coordinate gene regulation and other DNA transactions across spans in the genome that are larger than the separation between the binding sites.

The identification of regulatory targets of all TFs is critical for understanding the entire network of the genome regulation. The lac regulon of Escherichia coli K-12 W3110 is composed of the lacZYA operon and its repressor lacI gene, and has long been recognized as the seminal model of transcription regulation in bacteria with only one highly preferred target. After the Genomic SELEX screening in vitro of more than 200 transcription factors (TFs) from E. coli K-12, however, we found that most TFs regulate multiple target genes. With respect to the number of regulatory targets, a total of these 200 E. coli TFs form a hierarchy ranging from a single target to as many as 1000 targets. Here we focus a total of 13 single-target TFs, 9 known TFs (BetI, KdpE, LacI, MarR, NanR, RpiR, TorR, UlaR and UxuR) and 4 uncharacterized TFs (YagI, YbaO, YbiH and YeaM), altogether forming only a minor group of TFs in E. coli. These single-target TFs were classified into three groups based on their functional regulation.

The short 8-10 amino acid "hinge" sequence in lactose repressor (LacI), present in other LacI/GalR family members, links DNA and inducer-binding domains. Structural studies of full-length or truncated LacI-operator DNA complexes demonstrate insertion of the dimeric helical "hinge" structure at the center of the operator sequence. This association bends the DNA ∼40° and aligns flanking semi-symmetric DNA sites for optimal contact by the N-terminal helix-turn-helix (HtH) sequences within each dimer. In contrast, the hinge region remains unfolded when bound to nonspecific DNA sequences. To determine ability of the hinge helix alone to mediate DNA binding, we examined (i) binding of LacI variants with deletion of residues 1-50 to remove the HtH DNA binding domain or residues 1-58 to remove both HtH and hinge domains and (ii) binding of a synthetic peptide corresponding to the hinge sequence with a Val52Cys substitution that allows reversible dimer formation via a disulfide linkage. Binding affinity for DNA is orders of magnitude lower in the absence of the helix-turn-helix domain with its highly positive charge. LacI missing residues 1-50 binds to DNA with ∼4-fold greater affinity for operator than for nonspecific sequences with minimal impact of inducer presence; in contrast, LacI missing residues 1-58 exhibits no detectable affinity for DNA. In oxidized form, the dimeric hinge peptide alone binds to O1 and nonspecific DNA with similarly small difference in affinity; reduction to monomer diminished binding to both O1 and nonspecific targets. These results comport with recent reports regarding LacI hinge interaction with DNA sequences., (© 2018 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.)

Protein-mediated DNA looping is ubiquitous in chromatin organization and gene regulation, but to what extent supercoiling or nucleoid associated proteins promote looping is poorly understood. Using the lac repressor (LacI), a paradigmatic loop-mediating protein, we measured LacI-induced looping as a function of either supercoiling or the concentration of the HU protein, an abundant nucleoid protein in Escherichia coli. Negative supercoiling to physiological levels with magnetic tweezers easily drove the looping probability from 0 to 100% in single DNA molecules under slight tension that likely exists in vivo. In contrast, even saturating (micromolar) concentrations of HU could not raise the looping probability above 30% in similarly stretched DNA or 80% in DNA without tension. Negative supercoiling is required to induce significant looping of DNA under any appreciable tension.

Genetic switches must alternate between states whose probabilities are dependent on regulatory signals. Classical examples of transcriptional control in bacteria depend on repressive DNA loops anchored by proteins whose structures are sensitive to small molecule inducers or co-repressors. We are interested in exploiting these natural principles to engineer artificial switches for transcriptional control of bacterial genes. Here, we implement designed homodimeric DNA looping proteins ('Transcription Activator-Like Effector Dimers'; TALEDs) for this purpose in living bacteria. Using well-studied FKBP dimerization domains, we build switches that mimic regulatory characteristics of classical Escherichia coli lactose, galactose and tryptophan operon promoters, including induction or co-repression by small molecules. Engineered DNA looping using TALEDs is thus a new approach to tuning gene expression in bacteria. Similar principles may also be applicable for gene control in eukaryotes.

Vector-dependent gene overexpression typically relies on an efficient operon and sufficient RNA polymerases (RNAPs). The lac (lactose) operon is a paradigm of transcription control, and cyclic AMP receptor protein (CRP) is a global regulator capable of recruiting RNAPs. However, the gap between lac operon and CRP has not been well bridged. In this work, CRP was fused to lac repressor protein (lacI) to form an artificial transcription factor (ATF) with the expectation that when LacI acted on the lacO-positioned upstream of gene of interest, the LacI-tethered CRP would trap RNAPs and thus improve the expression of PuuC, an aldehyde dehydrogenase catalyzing 3-hydroxypropionaldehyde (3-HPA) to 3-hydroxypropionic acid (3-HP) in Klebsiella pneumoniae. As expected, SDS-PAGE and HPLC showed enhanced PuuC expression and 3-HP production, respectively, compared to the control strain without expressing chimeric protein LacI-CRP. Moreover, quantitative real-time PCR demonstrated increased transcription levels of both PuuC and RNAP coding genes. In shake-flask cultivation, the recombinant K. pneumoniae strain coexpressing LacI-CRP and PuuC produced 1.67-fold of 3-HP relative to the stain only overexpressing PuuC. In bioreactor cultivation, the strain coexpressing LacI-CRP and PuuC produced 35.1 g/L 3-HP, whereas the strain without expressing LacI-CRP generated only 9.8 g/L 3-HP. Overall, these results indicated that as an ATF, LacI-CRP significantly boosted PuuC expression and 3-HP production. We envision that LacI-CRP as a plug-and-play part can be used for regulating gene expression.

Significant, otherwise-unavailable information about mechanisms and transition states (TS) of protein folding and binding is obtained from solute effects on rate constants. Here we characterize TS for lac repressor(R)-lac operator(O) binding by analyzing effects of RO-stabilizing and RO-destabilizing solutes on association (ka) and dissociation (kd) rate constants. RO-destabilizing solutes (urea, KCl) reduce ka comparably (urea) or more than (KCl) they increase kd, demonstrating that they destabilize TS relative to reactants and RO, and that TS exhibits most of the Coulombic interactions between R and O. Strikingly, three solutes which stabilize RO by favoring burial/dehydration of amide oxygens and anionic phosphate oxygens all reduce kd without affecting ka significantly. The lack of stabilization of TS by these solutes indicates that O phosphates remain hydrated in TS and that TS preferentially buries aromatic carbons and amide nitrogens while leaving amide oxygens exposed. In our proposed mechanism, DNA-binding-domains (DBD) of R insert in major grooves of O pre-TS, forming most Coulombic interactions of RO and burying aromatic carbons. Nucleation of hinge helices creates TS, burying sidechain amide nitrogens. Post-TS, hinge helices assemble and the DBD-hinge helix-O-DNA module docks on core repressor, partially dehydrating phosphate oxygens and tightening all interfaces to form RO., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Previous investigations of the sensitivity of the lac repressor to high-hydrostatic pressure have led to varying conclusions. Here high-pressure solution NMR spectroscopy is used to provide an atomic level view of the pressure induced structural transition of the lactose repressor regulatory domain (LacI* RD) bound to the ligand IPTG. As the pressure is raised from ambient to 3kbar the native state of the protein is converted to a partially unfolded form. Estimates of rotational correlation times using transverse optimized relaxation indicates that a monomeric state is never reached and that the predominate form of the LacI* RD is dimeric throughout this pressure change. Spectral analysis suggests that the pressure-induced transition is localized and is associated with a volume change of approximately -115mlmol -1 and an average pressure dependent change in compressibility of approximately 30mlmol -1 kbar -1 . In addition, a subset of resonances emerge at high-pressures indicating the presence of a non-native but folded alternate state., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We report here a robust, tunable, and reversible transcription control system for endogenous genes. The REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci) employs enhanced lac repression and tet activation systems. With this approach, we show in mouse embryonic stem cells that endogenous Dnmt1 gene transcription could be up- or downregulated in a tunable, inducible, and reversible manner across nearly two orders of magnitude. Transcriptional repression of Dnmt1 by REMOTE-control was potent enough to cause embryonic lethality in mice, reminiscent of a genetic knockout of Dnmt1 and could substantially suppress intestinal polyp formation when applied to an ApcMin model. Binding by the enhanced lac repressor was sufficiently tight to allow strong attenuation of transcriptional elongation, even at operators located many kilobases downstream of the transcription start site and to produce invariably tight repression of all of the strong viral/mammalian promoters tested. Our approach of targeting tet transcriptional activators to the endogenous Dnmt1 promoter resulted in robust upregulation of this highly expressed housekeeping gene. Our system provides exquisite control of the level, timing, and cell-type specificity of endogenous gene expression, and the potency and versatility of the system will enable high resolution in vivo functional analyses., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We describe an electrochemical measurement technique that enables bioelectronic measurements of reporter proteins in living cells as an alternative to traditional optical fluorescence. Using electronically programmable microfluidics, the measurement is in turn used to control the concentration of an inducer input that regulates production of the protein from a genetic promoter. The resulting bioelectronic and microfluidic negative-feedback loop then serves to regulate the concentration of the protein in the cell. We show measurements wherein a user-programmable set-point precisely alters the protein concentration in the cell with feedback-loop parameters affecting the dynamics of the closed-loop response in a predictable fashion. Our work does not require expensive optical fluorescence measurement techniques that are prone to toxicity in chronic settings, sophisticated time-lapse microscopy, or bulky/expensive chemo-stat instrumentation for dynamic measurement and control of biomolecules in cells. Therefore, it may be useful in creating a: cheap, portable, chronic, dynamic, and precise all-electronic alternative for measurement and control of molecules in living cells.

Nuclear RNAs emerge as important factors to orchestrate the dynamic organization of the nucleus into functional subcompartments. By tethering RNAs to distinct genomic loci, RNA-dependent chromatin changes can be dissected by fluorescence microscopic analysis. Here we describe how this approach is implemented in mammalian cells. It involves two high-affinity protein-nucleic acid interactions that can be established with a number of different protein domains and DNA and RNA sequences. A prototypic system is described here in detail: It consists of the binding of MS2 bacteriophage coat protein to its RNA recognition sequence and the interaction between the bacterial LacI repressor protein to its target lacO operator DNA sequence. Via these interactions RNAs tagged with the MS2 recognition sequences can be recruited to a locus with integrated lacO repeats. By inducing RNA-chromatin binding a number of RNA-dependent activities can be dissected: (i) The RNA-induced compaction or decondensation of chromatin, (ii) identification of RNA-interacting chromatin modifiers that set epigenetic signals such as posttranslational histone modifications, and (iii) nuclear relocation of a genomic locus targeted by the tethered RNA. Thus, a variety of RNA-dependent activities can be evaluated with the MS2-LacI system, which are crucial for understanding how RNA shapes nuclear organization., (Copyright © 2017 Elsevier Inc. All rights reserved.)