Maximizing protein production by keeping cells at optimal secretory stress levels using real-time control approaches.

Optimizing the production of recombinant proteins is a problem of major industrial and pharmaceutical importance. Secretion of the protein by the host cell considerably simplifies downstream purification processes. However, for many proteins, this is also the limiting production step. Current solutions involve extensive engineering of the chassis cell to facilitate protein trafficking and limit protein degradation triggered by excessive secretion-associated stress. Here, we propose instead a regulation-based strategy in which induction is dynamically adjusted to an optimal strength based on the current stress level of the cells. Using a small collection of hard-to-secrete proteins, a bioreactor-based platform with automated cytometry measurements, and a systematic assay to quantify secreted protein levels, we demonstrate that the secretion sweet spot is indicated by the appearance of a subpopulation of cells that accumulate high amounts of proteins, decrease growth, and face significant stress, that is, experience a secretion burnout. In these cells, adaptations capabilities are overwhelmed by a too strong production. Using these notions, we show for a single-chain antibody variable fragment that secretion levels can be improved by 70% by dynamically keeping the cell population at optimal stress levels using real-time closed-loop control.

Low-cost anti-mycobacterial drug discovery using engineered E. coli.

Whole-cell screening for Mycobacterium tuberculosis (Mtb) inhibitors is complicated by the pathogen's slow growth and biocontainment requirements. Here we present a synthetic biology framework for assaying Mtb drug targets in engineered E. coli. We construct Target Essential Surrogate E. coli (TESEC) in which an essential metabolic enzyme is deleted and replaced with an Mtb-derived functional analog, linking bacterial growth to the activity of the target enzyme. High throughput screening of a TESEC model for Mtb alanine racemase (Alr) revealed benazepril as a targeted inhibitor, a result validated in whole-cell Mtb. In vitro biochemical assays indicated a noncompetitive mechanism unlike that of clinical Alr inhibitors. We establish the scalability of TESEC for drug discovery by characterizing TESEC strains for four additional targets.

Enhancing bioreactor arrays for automated measurements and reactive control with ReacSight.

Small-scale, low-cost bioreactors provide exquisite control of environmental parameters of microbial cultures over long durations. Their use is gaining popularity in quantitative systems and synthetic biology. However, existing setups are limited in their measurement capabilities. Here, we present ReacSight, a strategy to enhance bioreactor arrays for automated measurements and reactive experiment control. ReacSight leverages low-cost pipetting robots for sample collection, handling and loading, and provides a flexible instrument control architecture. We showcase ReacSight capabilities on three applications in yeast. First, we demonstrate real-time optogenetic control of gene expression. Second, we explore the impact of nutrient scarcity on fitness and cellular stress using competition assays. Third, we perform dynamic control of the composition of a two-strain consortium. We combine custom or chi.bio reactors with automated cytometry. To further illustrate ReacSight's genericity, we use it to enhance plate-readers with pipetting capabilities and perform repeated antibiotic treatments on a bacterial clinical isolate.

Enabling reactive microscopy with MicroMator.

Microscopy image analysis has recently made enormous progress both in terms of accuracy and speed thanks to machine learning methods and improved computational resources. This greatly facilitates the online adaptation of microscopy experimental plans using real-time information of the observed systems and their environments. Applications in which reactiveness is needed are multifarious. Here we report MicroMator, an open and flexible software for defining and driving reactive microscopy experiments. It provides a Python software environment and an extensible set of modules that greatly facilitate the definition of events with triggers and effects interacting with the experiment. We provide a pedagogic example performing dynamic adaptation of fluorescence illumination on bacteria, and demonstrate MicroMator's potential via two challenging case studies in yeast to single-cell control and single-cell recombination, both requiring real-time tracking and light targeting at the single-cell level.

P40 and P75 Are Singular Functional Muramidases Present in the Lactobacillus casei /paracasei/rhamnosus Taxon.

Lactobacillus casei and Lactobacillus rhamnosus proteins P40 and P75 belong to a large family of secreted cell wall proteins that contain a carboxy(C)-terminal CHAP or NlpC/P60 superfamily domains. In addition to their peptidoglycan hydrolases activity, proteins in this family are specific antigens of pathogens, frequently responsible of interactions with the host. L. rhamnosus GG and L. casei BL23 purified P40 and P75 proteins have antiapoptotic activity by inducing the EGF/Akt pathway. The aim of this work was to study the genetics, phylogeny and dissemination of this family of proteins in the genus Lactobacillus as well as their characteristics and likely function. The scrutiny of their DNA encoding sequences revealed the presence of minisatellite DNA in the P75 encoding gene of L. casei/paracasei strains (cmuB) with intraspecific indels that gave raise to four different alleles (cmuB1-4), which are exclusive of this species. Phylogenic analyses suggest that both proteins are present mainly in the L. casei and Lactobacillus sakei phylogenomic groups. A P40 ancestral gene was possibly present in the common ancestor of Enterococcaceae, Lactobacillaceae and Streptococcaceae. P75 is also present in L. casei and L. sakei groups, but its evolution is difficult to explain only by vertical transmission. Antibodies raised against the N-terminal regions of P40 and P75 improved their immunological detection in culture supernatants as they recognized almost exclusively proteins of L. casei/paracasei/rhamnosus strains, highlighting their structural similarity, that allowed to detect them in different fermented dairy products that contained probiotic L. casei strains. Purified P40 and P75 proteins showed no evident lytic activity but they complemented L. casei BL23 cmuA and cmuB defective mutants, respectively, thus proving that they actively participate in cell division.

Balancing a genetic toggle switch by real-time feedback control and periodic forcing.

Cybergenetics is a novel field of research aiming at remotely pilot cellular processes in real-time with to leverage the biotechnological potential of synthetic biology. Yet, the control of only a small number of genetic circuits has been tested so far. Here we investigate the control of multistable gene regulatory networks, which are ubiquitously found in nature and play critical roles in cell differentiation and decision-making. Using an in silico feedback control loop, we demonstrate that a bistable genetic toggle switch can be dynamically maintained near its unstable equilibrium position for extended periods of time. Importantly, we show that a direct method based on dual periodic forcing is sufficient to simultaneously maintain many cells in this undecided state. These findings pave the way for the control of more complex cell decision-making systems at both the single cell and the population levels, with vast fundamental and biotechnological applications.