Stochastic Neural Networks for Automatic Cell Tracking in Microscopy Image Sequences of Bacterial Colonies

Our work targets automated analysis to quantify the growth dynamics of a population of bacilliform bacteria. We propose an innovative approach to frame-sequence tracking of deformable-cell motion by the automated minimization of a new, specific cost functional. This minimization is implemented by dedicated Boltzmann machines (stochastic recurrent neural networks). Automated detection of cell divisions is handled similarly by successive minimizations of two cost functions, alternating the identification of children pairs and parent identification. We validate the proposed automatic cell tracking algorithm using (i) recordings of simulated cell colonies that closely mimic the growth dynamics of E. coli in microfluidic traps and (ii) real data. On a batch of 1100 simulated image frames, cell registration accuracies per frame ranged from 94.5% to 100%, with a high average. Our initial tests using experimental image sequences (i.e., real data) of E. coli colonies also yield convincing results, with a registration accuracy ranging from 90% to 100%

Majority sensing in synthetic microbial consortia

Designing distributed circuits that respond predictably to variation in bacterial populations remains difficult. Here the authors develop a two-strain circuit that senses and responds to the majority strain

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 <i>Escherichia coli</i>. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants that initiated translation within the PAS and GAF domains were discovered. Circularly permuted iRFPs retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a quantum yield similar to that of iRFPs but exhibited increased resistance to chemical denaturation, suggesting that the observed increase in the magnitude of the signal arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step toward the creation of near-infrared biosensors with expanded chemical sensing functions for <i>in vivo</i> imaging

Decaffeination and Measurement of Caffeine Content by Addicted <i>Escherichia coli</i> with a Refactored <i>N</i>‑Demethylation Operon from <i>Pseudomonas putida</i> 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 <i>Pseudomonas putida</i> CBB5 to function in <i>Escherichia coli</i>. In the process, we discovered that adding a glutathione <i>S</i>-transferase from <i>Janthinobacterium</i> sp. Marseille was necessary to achieve <i>N</i>₇-demethylation activity. <i>E. coli</i> cells with the synthetic operon degrade caffeine to the guanine precursor, xanthine. Cells deficient in <i>de novo</i> guanine biosynthesis that contain the refactored operon are ″addicted″ to caffeine: their growth density is limited by the availability of caffeine or other xanthines. We show that the addicted strain can be used as a biosensor to measure the caffeine content of common beverages. The synthetic <i>N</i>-demethylation operon could be useful for reclaiming nutrient-rich byproducts of coffee bean processing and for the cost-effective bioproduction of methylxanthine drugs