10 research outputs found
Riboswitch-Based Reversible Dual Color Sensor
Riboswitches
are RNA-based “sensors” that utilize
chemically induced structural changes in the 5′-untranslated
region of mRNA to regulate expression of downstream genes. Coupling
a specific riboswitch with a reporter gene system translates chemical
detection by the cell into a quantifiable reporter protein signal.
For the majority of reporter gene systems, the readout signal is only
expressed in the presence of the target analyte. This makes it difficult
to determine the viability and localization of the uninduced biosensor
when it is used for “real-word” applications. To address
this problem, we developed a dual-color reporter comprising elements
of the <i>E. coli</i> fimbriae phase variation system: recombinase
FimE controlled by a synthetic riboswitch and an invertible DNA segment
(<i>fimS</i>) containing a constitutively active promoter
placed between two fluorescent protein genes. Without an analyte,
the fluorescent reporter constitutively expressed green fluorescent
protein (GFPa1). Addition of the analyte initiated translation of <i>fimE</i> causing unidirectional inversion of the <i>fimS</i> segment and constitutive expression of red fluorescent protein (mKate2).
Thus, the sensor is always fluorescent, but its color is determined
by detection of a specific analyte. We demonstrate that the recombinase-based
dual-color reporter can be successfully applied to monitor the activation
of a theophylline synthetic riboswitch that was used as our model
system. To show the feasibility of the FimE recombinase-based system
to serve as a reporter for monitoring activation of multiple synthetic
riboswitches and, therefore, expand the applicability of the system,
we tested a number of previously developed synthetic riboswitches
responsive to different analytes. We show that the dual-color reporter
system can be successfully used to monitor activation of M6 and M6″
riboswitches responsive to ammeline and pyrimido[4,5-<i>d</i>]pyrimidine-2,4-diamine, respectively, and a 2,4,6-trinitrotoluene-responsive
riboswitch developed in this study. We also demonstrate that the system
can be reversed by HbiF recombinase-mediated <i>fimS</i> inversion to the initial state of the fluorescent reporter, creating
a resettable and reusable cell-based sensor
Development of a 2,4-Dinitrotoluene-Responsive Synthetic Riboswitch in <i>E. coli</i> Cells
Riboswitches are RNA sequences that regulate expression
of associated
downstream genes in response to the presence or absence of specific
small molecules. A novel riboswitch that activates protein translation
in <i>E. coli</i> cells in response to 2,4-dinitrotoluene
(DNT) has been engineered. A plasmid library was constructed by incorporation
of 30 degenerate bases between a previously described trinitrotoluene
aptamer and the ribosome binding site. Screening was performed by
placing the riboswitch library upstream of the Tobacco Etch Virus
(TEV) protease coding sequence in one plasmid; a second plasmid encoded
a FRET-based construct linked with a peptide containing the TEV protease
cleavage site. Addition of DNT to bacterial culture activated the
riboswitch, initiating translation of TEV protease. In turn, the protease
cleaved the linker in the FRET-based fusion protein, causing a change
in fluorescence. This new riboswitch exhibited a 10-fold increase
in fluorescence in the presence of 0.5 mM DNT compared to the system
without target
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octobre 19361936/10 (A3)-1936/10.Appartient à l’ensemble documentaire : RhoneAlp
Immobilization of Recombinant <i>E. coli</i> Cells in a Bacterial Cellulose–Silk Composite Matrix To Preserve Biological Function
Strategies for the encapsulation
of cells for the design of cell-based
sensors require efficient immobilization procedures while preserving
biological activity of the reporter cells. Here, we introduce an immobilization
technique that relies upon the symbiotic relationship between two
bacterial strains: cellulose-producing <i>Gluconacetobacter xylinus</i> cells; and recombinant <i>Escherichia coli</i> cells harboring
recombinase-based dual-color synthetic riboswitch (RS), as a model
for cell-based sensor. Following sequential coculturing of recombinant
cells in the cellulose matrix, final immobilization of <i>E.
coli</i> cells was completed after reconstituted silk fibroin
(SF) protein was added to a “living membrane” generating
the composite bacterial cellulose-silk fibroin (BC-SF) scaffold. By
controlling incubation parameters for both types of cells, as well
as the conformations in SF secondary structure, a variety of robust
composite scaffolds were prepared ranging from opaque to transparent.
The properties of the scaffolds were compared in terms of porosity,
water capacity, distribution of recombinant cells within the scaffolds
matrix, onset of cells activation, and ability to protect recombinant
function of cells against UV irradiation. The closer-fitted microstructure
of transparent BC-SF scaffolds resulted in leakage-free encapsulation
of recombinant cells with preserved RS function because of a combination
of several parameters that closely matched properties of a biofilm
environment. Along with proper elasticity, fine porosity, capacity
to retain the water, and ability of SF to absorb UV light, the composite
hydrogel material provided necessary conditions to form confined cell
colonies that modified cell metabolism and enhanced cell resilience
to the stresses induced by encapsulation
Optimization of a Paper-Based ELISA for a Human Performance Biomarker
Monitoring aspects of human performance
during various activities
has recently become a highly investigated research area. Many new
commercial products are available now to monitor human physical activity
or responses while performing activities ranging from playing sports,
to driving, and even sleeping. However, monitoring cognitive performance
biomarkers, such as neuropeptides, is still an emerging field due
to the complicated sample collection and processing, as well as the
need for a clinical lab to perform analysis. Enzyme-linked immunosorbent
assays (ELISAs) provide specific detection of biomolecules with high
sensitivity (picomolar concentrations). Even with the advantage of
high sensitivity, most ELISAs need to be performed in a laboratory
setting and require around 6 h to complete. Transitioning this assay
to a platform where it reduces cost, shortens assay time, and is able
to be performed outside a lab is invaluable. Recently developed paper
diagnostics provide an inexpensive platform on which to perform ELISAs;
however, the major limiting factor for moving out of the laboratory
environment is the measurement and analysis instrumentation. Using
something as simple as a digital camera or camera-enabled Windows-
or Android-based tablets, we are able to image paper-based ELISAs
(P-ELISAs), perform image analysis, and produce response curves with
high correlation to target biomolecule concentration in the 10 pM
range. Neuropeptide Y detection was performed. Additionally, silver
enhancement of Au NPs conjugated with IgG antibodies showed a concentration-dependent
response to IgG, thus eliminating the need for an enzyme–substrate
system. Automated image analysis and quantification of antigen concentrations
are able to be performed on Windows- and Android-based mobile platforms
Silk Macromolecules with Amino Acid–Poly(Ethylene Glycol) Grafts for Controlling Layer-by-Layer Encapsulation and Aggregation of Recombinant Bacterial Cells
This study introduces double-brush designs of functionalized silk polyelectrolytes based upon regenerated silk fibroin (SF), which is modified with poly-l-lysine (SF-PLL), poly-l-glutamic acid (SF-PGA), and poly(ethylene glycol) (PEG) side chains with different grafting architecture and variable amino acid-PEG graft composition for cell encapsulation. The molecular weight of poly amino acids (length of side chains), molecular weight and degree of PEG grafting (<i>D</i>) were varied in order to assess the formation of cytocompatible and robust layer-by-layer (LbL) shells on two types of bacterial cells (Gram-negative and Gram-positive bacteria). We observed that shells assembled with charged polycationic amino acids adversely effected the properties of microbial cells while promoting the formation of large cell aggregates. In contrast, hydrogen-bonded shells with high PEG grafting density were the most cytocompatible, while promoting formation of stable colloidal suspensions of individual cell encapsulates. The stability to degradation of silk shells (under standard cell incubation procedure) was related to the intrinsic properties of thermodynamic bonding forces, with shells based on electrostatic interactions having stronger resistance to deterioration compared to pure hydrogen-bonded silk shells. By optimizing the charge density of silk polyelectrolytes brushes, as well as the length and the degree of PEG side grafts, robust and cytocompatible cell coatings were engineered that can control aggregation of cells for biosensor devices and other potential biomedical applications
Structured DNA Aptamer Interactions with Gold Nanoparticles
DNA
aptamers that bind biomolecular targets are of interest as
the recognition element in colorimetric sensors based on gold nanoparticles
(AuNP), where sensor functionality is related to changes in AuNP colloidal
stability upon target binding. In order to understand the role of
target binding on DNA–AuNP colloidal stability, we have used
high-resolution NMR to characterize the interactions of the 36 nucleotide
cocaine-binding aptamer (MN4) and related aptamers with AuNPs, cocaine,
and cocaine metabolites. Changes in the aptamer imino proton NMR spectra
with low (20 nM) concentrations of AuNP show that the aptamers undergo
fast-exchange adsorption on the nanoparticle surface. An analysis
of the spectral changes and the comparison with modified MN4 aptamers
shows that the AuNP binding domain is localized on stem two of the
three-stemmed aptamer. The identification of an AuNP recognition domain
allows for the incorporation of AuNP binding functionality into a
wide variety of aptamers. AuNP-induced spectral changes are not observed
for the aptamer–AuNP mixtures in the presence of cocaine, demonstrating
that aptamer absorption on the AuNP surface is modulated by aptamer–target
interactions. The data also show that the DNA–AuNP interactions
and sensor functionality are critically dependent on aptamer folding
Bacterial Sunscreen: Layer-by-Layer Deposition of UV-Absorbing Polymers on Whole-Cell Biosensors
UV-protective coatings on live bacterial cells were created
from
the assembly of cationic and UV-absorbing anionic polyelectrolytes
using layer-by-layer (LbL) methodology. A cationic polymer (polyallylamine)
and three different anionic polymers with varying absorbance in the
UV range (poly(vinyl sulfate), poly(4-styrenesulfonic acid), and humic
acid) were used to encapsulate Escherichia coli cells with two different green fluorescent protein (GFP) expression
systems: constitutive expression of a UV-excitable GFP (GFPuv) and
regulated expression of the intensely fluorescent GFP from amphioxus
(GFPa1) through a theophylline-inducible riboswitch. Riboswitches
activate protein expression after specific ligand–RNA binding
events. Hence, they operate as a cellular biosensor that will activate
reporter protein synthesis after exposure to a ligand target. E. coli cells coated with UV-absorbing polymers demonstrated
enhanced protection of GFP stability, metabolic activity, and viability
after prolonged exposure to radiation from a germicidal lamp. The
results show the effectiveness of LbL coatings to provide UV protection
to living cells for biotechnological applications
Influence of Silica Matrix Composition and Functional Component Additives on the Bioactivity and Viability of Encapsulated Living Cells
The remarkable impact encapsulation
matrix chemistry can have on
the bioactivity and viability of integrated living cells is reported.
Two silica chemistries (aqueous silicate and alkoxysilane), and a
functional component additive (glycerol), are employed to generate
three distinct silica matrices. These matrices are used to encapsulate
living <i>E. coli</i> cells engineered with a synthetic
riboswitch for cell-based biosensing. Following encapsulation, membrane
integrity, reproductive capability, and riboswitch-based protein expression
levels and rates are measured over a 5 week period. Striking differences
in <i>E. coli</i> bioactivity, viability, and biosensing
performance are observed for cells encapsulated within the different
matrices. <i>E. coli</i> cells encapsulated for 35 days
in aqueous silicate-based (AqS) matrices showed relatively low membrane
integrity, but high reproductive capability in comparison to cells
encapsulated in glycerol containing sodium silicate-based (AqS + g)
and alkoxysilane-based (PGS) gels. Further, cells in sodium silicate-based
matrices showed increasing fluorescence output over time, resulting
in a 1.8-fold higher fluorescence level, and a faster expression rate,
over cells free in solution. This unusual and unique combination of
biological properties demonstrates that careful design of the encapsulation
matrix chemistry can improve functionality of the biocomposite material,
and result in new and unexpected physiological states