Whole cell biosensors are genetic systems that link the presence of a chemical, or other stimulus, to a user-defined gene expression output for applications in sensing and control. However, the gene expression level of biosensor regulatory components required for optimal performance is nonintuitive, and classical iterative approaches do not efficiently explore multidimensional experimental space. To overcome these challenges, we used a design of experiments (DoE) methodology to efficiently map gene expression levels and provide biosensors with enhanced performance. This methodology was applied to two biosensors that respond to catabolic breakdown products of lignin biomass, protocatechuic acid and ferulic acid. Utilizing DoE we systematically modified biosensor dose–response behavior by increasing the maximum signal output (up to 30-fold increase), improving dynamic range (>500-fold), expanding the sensing range (∼4-orders of magnitude), increasing sensitivity (by >1500-fold), and modulated the slope of the curve to afford biosensors designs with both digital and analogue dose–response behavior. This DoE method shows promise for the optimization of regulatory systems and metabolic pathways constructed from novel, poorly characterized parts.
To facilitate the screening of chemical, enzymatic, and cellular processes to degrade and valorize plant biomass a whole cell biosensor was developed to detect lignin-derived substrates.
Background:Genetically encoded biosensors are useful tools for the detection of metabolites and industrially valuable molecules, and present many potential applications in biotechnology and biomedicine. However, the most common approach to develop biosensors relies on employing a limited set of naturally occurring allosteric transcript factors (aTFs). Therefore, altering the substrate specificity of aTFs towards the detection of new effectors is an important goal. Results:Here, the PcaV repressor, a member of the MarR aTF family, was used to develop a biosensor for the detection of hydroxyl-substituted benzoic acids, including protocatechuic acid (PCA). The PCA biosensor was further subjected to directed evolution to alter its substrate specificity towards vanillin and other closely related aromatic aldehydes, to generate the Van2 biosensor. Substrate recognition of Van2 was explored in vitro using a range of biochemical and biophysical analyses, and extensive in vivo genetic-phenotypic analysis was performed to determine the role of each amino acid change upon biosensor performance. Conclusions:This is the first study to report directed evolution of a member of the MarR aTF family, and demonstrates the plasticity of the PCA biosensor by altering its substrate specificity to generate a biosensor for aromatic aldehydes.
BackgroundTranscription factor-based biosensors are useful tools for the detection of metabolites and industrially valuable molecules, and present many potential applications in biotechnology and biomedicine. However, the most common approach to develop biosensors relies on employing a limited set of naturally occurring allosteric transcription factors (aTFs). Therefore, altering the ligand specificity of aTFs towards the detection of new effectors is an important goal.ResultsHere, the PcaV repressor, a member of the MarR aTF family, was used to develop a biosensor for the detection of hydroxyl-substituted benzoic acids, including protocatechuic acid (PCA). The PCA biosensor was further subjected to directed evolution to alter its ligand specificity towards vanillin and other closely related aromatic aldehydes, to generate the Van2 biosensor. Ligand recognition of Van2 was explored in vitro using a range of biochemical and biophysical analyses, and extensive in vivo genetic-phenotypic analysis was performed to determine the role of each amino acid change upon biosensor performance.ConclusionsThis is the first study to report directed evolution of a member of the MarR aTF family, and demonstrates the plasticity of the PCA biosensor by altering its ligand specificity to generate a biosensor for aromatic aldehydes.
Visceral leishmaniasis (VL) is a major public health issue reported as the second illness in mortality among all tropical diseases. Clinical trials have shown that protection against VL is associated with robust T cell responses, especially those producing IFN-γ. The Leishmania amastigote 2 (A2) protein has been repeatedly described as immunogenic and protective against VL in different animal models; it is recognized by human T cells, and it is also commercially available in a vaccine formulation containing saponin against canine VL. Moving toward a more appropriate formulation for human vaccination, here, we tested a new optimized version of the recombinant protein (rA2), designed for Escherichia coli expression, in combination with adjuvants that have been approved for human use. Moreover, aiming at improving the cellular immune response triggered by rA2, we generated a recombinant live vaccine vector using Trypanosoma cruzi CL-14 non-virulent strain, named CL-14 A2. Mice immunized with respective rA2, adsorbed in Alum/CpG B297, a TLR9 agonist recognized by mice and human homologs, or with the recombinant CL-14 A2 parasites through homologous prime-boost protocol, were evaluated for antigen-specific immune responses and protection against Leishmania infantum promastigote challenge. Immunization with the new rA2/Alum/CpG formulations and CL-14 A2 transgenic vectors elicited stronger cellular immune responses than control groups, as shown by increased levels of IFN-γ, conferring protection against L. infantum challenge. Interestingly, the use of the wild-type CL-14 alone was enough to boost immunity and confer protection, confirming the previously reported immunogenic potential of this strain. Together, these results support the success of both the newly designed rA2 antigen and the ability of T. cruzi CL-14 to induce strong T cell-mediated immune responses against VL in animal models when used as a live vaccine vector. In conclusion, the vaccination strategies explored here reveal promising alternatives for the development of new rA2 vaccine formulations to be translated human clinical trials.
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