19 research outputs found
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><sub>7</sub>-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