Evaluating the predictive power of combined gene expression dynamics from single cells on antibiotic survival.

Heteroresistance can allow otherwise drug-susceptible bacteria to survive and resume growth after antibiotic exposure. This temporary form of antibiotic tolerance can be caused by the upregulation of stress response genes or a decrease in cell growth rate. However, it is not clear how expression of multiple genes contributes to the tolerance phenotype.

Evaluation of Choudhary et al.: Single-cell gene expression dynamics in the E. coli oxidative stress response network.

One snapshot of the peer review process for "The master regulator OxyR orchestrates bacterial oxidative stress response genes in space and time" (Choudhary et al., 2024)..

Tunable Dynamics in a Multistrain Transcriptional Pulse Generator.

One challenge in synthetic biology is the tuning of regulatory components within gene circuits to elicit a specific behavior. This challenge becomes more difficult in synthetic microbial consortia since each strain's circuit must function at the intracellular level and their combination must operate at the population level. Here we demonstrate that circuit dynamics can be tuned in synthetic consortia through the manipulation of strain fractions within the community.

Indirect Enrichment of Desirable, but Less Fit Phenotypes, from a Synthetic Microbial Community Using Microdroplet Confinement.

Spatial structure within microbial communities can provide nearly limitless opportunities for social interactions and are an important driver for evolution. As metabolites are often molecular signals, metabolite diffusion within microbial communities can affect the composition and dynamics of the community in a manner that can be challenging to deconstruct. We used encapsulation of a synthetic microbial community within microdroplets to investigate the effects of spatial structure and metabolite diffusion on population dynamics and to examine the effects of cheating by one member of the community.

Indirect enrichment of desirable, but less fit phenotypes, from a synthetic microbial community using microdroplet confinement.

Spatial structure within microbial communities can provide nearly limitless opportunities for social interactions and are an important driver for evolution. As metabolites are often molecular signals, metabolite diffusion within microbial communities can affect the composition and dynamics of the community in a manner that can be challenging to deconstruct. We used encapsulation of a synthetic microbial community within microdroplets to investigate the effects of spatial structure and metabolite diffusion on population dynamics and to examine the effects of cheating by one member of the community.

Advances in linking single-cell bacterial stress response to population-level survival.

Stress response mechanisms can allow bacteria to survive a myriad of challenges, including nutrient changes, antibiotic encounters, and antagonistic interactions with other microbes. Expression of these stress response pathways, in addition to other cell features such as growth rate and metabolic state, can be heterogeneous across cells and over time. Collectively, these single-cell-level phenotypes contribute to an overall population-level response to stress.

DeLTA 2.0: A deep learning pipeline for quantifying single-cell spatial and temporal dynamics.

Improvements in microscopy software and hardware have dramatically increased the pace of image acquisition, making analysis a major bottleneck in generating quantitative, single-cell data. Although tools for segmenting and tracking bacteria within time-lapse images exist, most require human input, are specialized to the experimental set up, or lack accuracy. Here, we introduce DeLTA 2.

Majority sensing in synthetic microbial consortia.

As synthetic biocircuits become more complex, distributing computations within multi-strain microbial consortia becomes increasingly beneficial. However, designing distributed circuits that respond predictably to variation in consortium composition remains a challenge. Here we develop a two-strain gene circuit that senses and responds to which strain is in the majority.

Long-range temporal coordination of gene expression in synthetic microbial consortia.

Synthetic microbial consortia have an advantage over isogenic synthetic microbes because they can apportion biochemical and regulatory tasks among the strains. However, it is difficult to coordinate gene expression in spatially extended consortia because the range of signaling molecules is limited by diffusion. Here, we show that spatio-temporal coordination of gene expression can be achieved even when the spatial extent of the consortium is much greater than the diffusion distance of the signaling molecules.

Spatiotemporal Dynamics of Synthetic Microbial Consortia in Microfluidic Devices.

Synthetic microbial consortia consist of two or more engineered strains that grow together and share the same resources. When intercellular signaling pathways are included in the engineered strains, close proximity of the microbes can generate complex dynamic behaviors that are difficult to obtain using a single strain. However, when a consortium is not cultured in a well-mixed environment the constituent strains passively compete for space as they grow and divide, complicating cell-cell signaling.

Tolerance of a Knotted Near-Infrared Fluorescent Protein to Random Circular Permutation.

Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFPs to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified 27 circularly permuted iRFPs that display biliverdin-dependent fluorescence in Escherichia coli.

Expanded Genetic Codes Create New Mutational Routes to Rifampicin Resistance in Escherichia coli.

Until recently, evolutionary questions surrounding the nature of the genetic code have been mostly limited to the realm of conjecture, modeling, and simulation due to the difficulty of altering this fundamental property of living organisms. Concerted genome and protein engineering efforts now make it possible to experimentally study the impact of alternative genetic codes on the evolution of biological systems. We explored how Escherichia coli strains that incorporate a 21st nonstandard amino acid (nsAA) at the recoded amber (TAG) stop codon evolve resistance to the antibiotic rifampicin.

The case for decoupling assembly and submission standards to maintain a more flexible registry of biological parts.

The Registry of Standard Biological Parts only accepts genetic parts compatible with the RFC 10 BioBrick format. This combined assembly and submission standard requires that four unique restriction enzyme sites must not occur in the DNA sequence encoding a part. We present evidence that this requirement places a nontrivial burden on iGEM teams developing large and novel parts.

Decaffeination and measurement of caffeine content by addicted Escherichia coli with a refactored N-demethylation operon from Pseudomonas putida CBB5.

The widespread use of caffeine (1,3,7-trimethylxanthine) and other methylxanthines in beverages and pharmaceuticals has led to significant environmental pollution. We have developed a portable caffeine degradation operon by refactoring the alkylxanthine degradation (Alx) gene cluster from Pseudomonas putida CBB5 to function in Escherichia coli. In the process, we discovered that adding a glutathione S-transferase from Janthinobacterium sp.