Engineering and application of LacI mutants with stringent expressions.

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 Plac /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).

A tuneable genetic switch for tight control of tac promoters in Escherichia coli boosts expression of synthetic injectisomes.

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 (LacIW220F ) with higher activity. The 3R switch was optimized with different protein translation and degradation signals that control the levels of LacIW220F . 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.

Imaging of genomic loci in Trypanosoma brucei using an optimised LacO-LacI system.

Visualisation of genomic loci by microscopy is essential for understanding nuclear organisation, particularly at the single cell level. One powerful technique for studying the positioning of genomic loci is through the Lac Operator-Lac Repressor (LacO-LacI) system, in which LacO repeats introduced into a specific genomic locus can be visualised through expression of a LacI-protein fused to a fluorescent tag. First utilised in Trypanosoma brucei over 20 years ago, we have now optimised this system with short, stabilised LacO repeats of less than 2 kb paired with a constitutively expressed mNeongreen::LacI fusion protein to facilitate visualisation of genomic loci. We demonstrate the compatibility of this system with super-resolution microscopy and propose its suitability for multiplexing with inducible RNAi or protein over expression which will allow analysis of nuclear organisation after perturbation of gene expression.

Genetic switching by the Lac repressor is based on two-state Monod-Wyman-Changeux allostery.

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.

Silencing Transcription from an Influenza Reverse Genetics Plasmid in E. coli Enhances Gene Stability.

Reverse genetics (RG) systems have been instrumental for determining the molecular aspects of viral replication, pathogenesis, and for the development of therapeutics. Here, we demonstrate that genes encoding the influenza surface antigens hemagglutinin and neuraminidase have varying stability when cloned into a common RG plasmid and transformed into Escherichia coli. Using GFP as a reporter, we demonstrate that E. coli expresses the target genes in the RG plasmid at low levels. Incorporating lac operators or a transcriptional terminator into the plasmid reduced expression and stabilized the viral genes to varying degrees. Sandwiching the viral gene between two lac operators provided the largest contribution to stability and we confirmed the stabilization is Lac repressor-dependent and crucial for subsequent plasmid propagations in E. coli. Viruses rescued from the lac operator-stabilized plasmid displayed similar kinetics and titers to the original plasmid in two different viral backbones. Together, these results indicate that silencing transcription from the plasmid in E. coli helps to maintain the correct influenza gene sequence and that the lac operator addition does not impair virus production. It is envisaged that sandwiching DNA segments between lac operators can be used for reducing DNA segment instability in any plasmid that is propagated in E. coli which express the Lac repressor.

Testing the Effect of Histone Acetyltransferases on Local Chromatin Compaction.

Experiments determining the chromatin association of histone acetylases (HATs) and deacetylases (HDACs) at the genome-wide level provide precise maps of locus occupancy, but do not allow conclusions on the functional consequences of this locus-specific enrichment. Here we describe a protocol that allows tethering of HATs or HDACs to specific genomic loci upon fusion with a fluorescent protein and a DNA-binding protein such as the E. coli Lac repressor (LacI), which binds to genomically inserted lac operon sequences (lacO) via DNA/protein interactions. Integration of these lacO sequences into a genomic region of interest allows to monitor the functional consequences of HAT/HDAC targeting on chromatin (de)compaction, histone modification, and interaction with other proteins by quantitative light microscopy, as described here. As DNA-binding of LacI can be tightly controlled by the addition of galactose-derivatives, this method also allows to monitor the effects of locus-specific recruitment in a time-resolved manner.

Robustness of DNA looping across multiple cell divisions in individual bacteria.

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.

Positive supercoiling favors transcription elongation through lac repressor-mediated DNA loops.

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.

Switching off: The phenotypic transition to the uninduced state of the lactose uptake pathway.

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.

Modular and Single-Cell Sensors of Bacterial Ser/Thr Kinase Activity.

At the single-cell level, protein kinase activity is typically inferred from downstream transcriptional reporters. However, promoters are often coregulated by several pathways, making the activity of a specific kinase difficult to deconvolve. Here, we present modular, direct, and specific sensors of bacterial kinase activity, including FRET-based sensors, as well as a synthetic transcription factor based on the lactose repressor (LacI) that has been engineered to respond to phosphorylation. We demonstrate the utility of these sensors in measuring the activity of PrkC, a conserved bacterial Ser/Thr kinase, in different growth conditions from single cells to colonies. We also show that PrkC activity increases in response to a cell-wall active antibiotic that blocks the late steps in peptidoglycan synthesis (cefotaxime), but not the early steps (fosfomycin). These sensors have a modular design that should generalize to other bacterial signaling systems in the future.

Biological innovation in the functional landscape of a model regulator, or the lactose operon repressor.

The operon model was proposed six decades ago. And yet, despite all this time, the lactose operon repressor, LacI, remains a subject of major interest. While it is well established that LacI can exist in two functional forms, one that renders the operon inactive via binding of LacI to DNA and another, bound to an inducer that does not allow repression, how it switches from one to the other is still not well understood. The construction of a library of several tens of thousands of LacI mutants has revealed some unexpected features. In particular, the transition implemented in some of them reveals a new type of transcription regulation: band-pass (OFF/ON/OFF) and band-stop (ON/OFF/ON) filters. This makes it natural to think that it is the network of hydrogen bonds associated with the water bound to the molecule that allows the remote interconnection between the binding site to an inducer molecule and the one that binds it to the DNA.

Le modèle de l'opéron a été proposé il y a six décennies. Et pourtant, malgré tout ce temps passé, le répresseur de l'opéron lactose, LacI, reste un sujet d'intérêt majeur. S'il est bien établi que LacI peut exister sous deux formes fonctionnelles, l'une qui rend inactif l'opéron via la liaison de LacI à l'ADN et l'autre, liée à un inducteur qui ne permet pas cette répression, la façon dont il passe de l'une à l'autre n'est toujours pas bien comprise. La construction d'une bibliothèque de plusieurs dizaines de milliers de mutants de LacI a mis au jour des caractéristiques inattendues. En particulier la transition mise en œuvre dans certains d'entre eux fait émerger un nouveau type de régulation de la transcription : filtre à bande passante (INACTIF/ACTIF/INACTIF) et filtre à bande d'arrêt (ACTIF/INACTIF/ACTIF). Il est naturel de penser que c'est le réseau des liaisons hydrogène associées à l'eau liée à la molécule qui permet l'interconnexion à distance entre le site de liaison à une molécule inductrice et celui qui le lie à l'ADN.

Rheostat functional outcomes occur when substitutions are introduced at nonconserved positions that diverge with speciation.

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.

A cleavage-based surrogate reporter for the evaluation of CRISPR-Cas9 cleavage efficiency.

CRISPR-Cas9 is a powerful tool for genome engineering, but its efficiency largely depends on guide RNA (gRNA). There are multiple methods available to evaluate the efficiency of gRNAs, including the T7E1 assay, surveyor nuclease assay, deep sequencing, and surrogate reporter systems. In the present study, we developed a cleavage-based surrogate that we have named the LacI-reporter to evaluate gRNA cleavage efficiency. The LacI repressor, under the control of the EF-1α promoter, represses luciferase or EGFP reporter expression by binding to the lac operator. Upon CRISPR-Cas9 cleavage at a target site located between the EF-1α promoter and the lacI gene, repressor expression is disrupted, thereby triggering luciferase or EGFP expression. Using this system, we can quantitate gRNA cleavage efficiency by assessing luciferase activity or EGFP expression. We found a strong positive correlation between the cleavage efficiency of gRNAs measured using this reporter and mutation frequency, measured using surveyor and deep sequencing. The genome-editing efficiency of gRNAs was validated in human liver organoids. Our LacI-reporter system provides a useful tool to select efficient gRNAs for genome editing.

Deep representation learning improves prediction of LacI-mediated transcriptional repression.

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.

Historical Contingency Causes Divergence in Adaptive Expression of the lac Operon.

Populations of Escherichia coli selected in constant and fluctuating environments containing lactose often adapt by substituting mutations in the lacI repressor that cause constitutive expression of the lac operon. These mutations occur at a high rate and provide a significant benefit. Despite this, eight of 24 populations evolved for 8,000 generations in environments containing lactose contained no detectable repressor mutations. We report here on the basis of this observation. We find that, given relevant mutation rates, repressor mutations are expected to have fixed in all evolved populations if they had maintained the same fitness effect they confer when introduced to the ancestor. In fact, reconstruction experiments demonstrate that repressor mutations have become neutral or deleterious in those populations in which they were not detectable. Populations not fixing repressor mutations nevertheless reached the same fitness as those that did fix them, indicating that they followed an alternative evolutionary path that made redundant the potential benefit of the repressor mutation, but involved unique mutations of equivalent benefit. We identify a mutation occurring in the promoter region of the uspB gene as a candidate for influencing the selective choice between these paths. Our results detail an example of historical contingency leading to divergent evolutionary outcomes.

The loopometer: a quantitative in vivo assay for DNA-looping proteins.

Proteins that can bring together separate DNA sites, either on the same or on different DNA molecules, are critical for a variety of DNA-based processes. However, there are no general and technically simple assays to detect proteins capable of DNA looping in vivo nor to quantitate their in vivo looping efficiency. Here, we develop a quantitative in vivo assay for DNA-looping proteins in Escherichia coli that requires only basic DNA cloning techniques and a LacZ assay. The assay is based on loop assistance, where two binding sites for the candidate looping protein are inserted internally to a pair of operators for the E. coli LacI repressor. DNA looping between the sites shortens the effective distance between the lac operators, increasing LacI looping and strengthening its repression of a lacZ reporter gene. Analysis based on a general model for loop assistance enables quantitation of the strength of looping conferred by the protein and its binding sites. We use this 'loopometer' assay to measure DNA looping for a variety of bacterial and phage proteins.

Programmable gene regulation for metabolic engineering using decoy transcription factor binding sites.

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.

Substitutions at Nonconserved Rheostat Positions Modulate Function by Rewiring Long-Range, Dynamic Interactions.

Amino acid substitutions at nonconserved protein positions can have noncanonical and "long-distance" outcomes on protein function. Such outcomes might arise from changes in the internal protein communication network, which is often accompanied by changes in structural flexibility. To test this, we calculated flexibilities and dynamic coupling for positions in the linker region of the lactose repressor protein. This region contains nonconserved positions for which substitutions alter DNA-binding affinity. We first chose to study 11 substitutions at position 52. In computations, substitutions showed long-range effects on flexibilities of DNA-binding positions, and the degree of flexibility change correlated with experimentally measured changes in DNA binding. Substitutions also altered dynamic coupling to DNA-binding positions in a manner that captured other experimentally determined functional changes. Next, we broadened calculations to consider the dynamic coupling between 17 linker positions and the DNA-binding domain. Experimentally, these linker positions exhibited a wide range of substitution outcomes: Four conserved positions tolerated hardly any substitutions ("toggle"), ten nonconserved positions showed progressive changes from a range of substitutions ("rheostat"), and three nonconserved positions tolerated almost all substitutions ("neutral"). In computations with wild-type lactose repressor protein, the dynamic couplings between the DNA-binding domain and these linker positions showed varied degrees of asymmetry that correlated with the observed toggle/rheostat/neutral substitution outcomes. Thus, we propose that long-range and noncanonical substitutions outcomes at nonconserved positions arise from rewiring long-range communication among functionally important positions. Such calculations might enable predictions for substitution outcomes at a range of nonconserved positions.

Characterization of Gene Repression by Designed Transcription Activator-like Effector Dimer Proteins.

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.

Engineered signal-coupled inducible promoters: measuring the apparent RNA-polymerase resource budget.

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.