Autoantibody detection by direct counting of antigen-displayed yeast cells.

In this study, we report a new immunoassay platform based on yeast surface display technology for detection of autoantibodies involved in autoimmune diseases, e.g., systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS). The autoantigens of Ro52/SSA epitope and SmD were chosen to be displayed on the yeast surface with their respective antibodies as the analytes. By using magnetic beads modified with protein G, yeast cells bound with specific target antibody can be captured. The amount of analytes could be determined by counting the number of fluorescent yeast cells captured in a magnetic field. The platform showed promising results in the detection of SLE autoantibodies with high sensitivity and multiplex detection capability over the traditional approaches.

Yeast surface display-based microfluidic immunoassay.

In this paper, we present a new microfluidic immunoassay platform, which is based on the synergistic combination of the yeast surface display (YSD) technique and the microfluidic technology. Utilizing the YSD technique, antigens specific to the target antibody are displayed on the surface of engineered yeast cells with intracellular fluorescent proteins. The displayed antigens are then used for the detection of the target antibody, with the yeast cells as fluorescent labels. Multiplex immunoassay can be readily realized by using yeast cells expressing different intracellular fluorescent proteins to display different antigens. The implementation of this YSD-based immunoassay on the microfluidic platform eliminates the need for the bulky, complex and expensive flow cytometer. To improve the detection sensitivity and to eliminate the need for pumping, a functionalized micro pillar array (MPA) is incorporated in the microfluidic chip, resulting in a detection limit of 5 ng/mL (or 1 ng in terms of amount) and enhanced compatibility with practical applications such as clinical biopsy. This new platform has a high potential to be integrated into microfluidic detection systems to enable portable diagnostics in the future.

New immunoassay platform utilizing yeast surface display and direct cell counting.

In this study, we report a new immunoassay platform using yeast cell surface display. This method holds promise for very low limit of detection (LOD) and is suitable for 2-Plex antibody recognition. Instead of adopting a conventional enzyme linked immunosorbent assay (ELISA) protocol by detecting the enzymatic activities or other physicochemical properties of the labeled analytes, this approach determines the quantity of an antibody analyte by directly counting the amount of "modified" yeast cells bound with antibody on the cell surface. c-myc and hemagglutinin (HA) tags were employed as an epitope model to demonstrate our approach. This yeast surface display based cell counting immunoassay (abbreviated as YSD-CCI) for anti-c-myc has a detection limit of 0.2 ng/mL, which is about 80 times higher than that of a conventional yeast ELISA under a similar condition. Moreover, the YSD-CCI's capability for 2-Plex antibody detection was demonstrated by simultaneous detection of anti-c-myc and anti-HA using engineered yeast cells expressing intracellular enhanced green fluorescent protein (EGFP) and mCherry, respectively. This proof-of-concept study paves the way for a new ultrasensitive multiplexed immunoassay method for diagnostic applications.

Microfluidics and microbial engineering.

The combination of microbial engineering and microfluidics is synergistic in nature. For example, microfluidics is benefiting from the outcome of microbial engineering and many reported point-of-care microfluidic devices employ engineered microbes as functional parts for the microsystems. In addition, microbial engineering is facilitated by various microfluidic techniques, due to their inherent strength in high-throughput screening and miniaturization. In this review article, we firstly examine the applications of engineered microbes for toxicity detection, biosensing, and motion generation in microfluidic platforms. Secondly, we look into how microfluidic technologies facilitate the upstream and downstream processes of microbial engineering, including DNA recombination, transformation, target microbe selection, mutant characterization, and microbial function analysis. Thirdly, we highlight an emerging concept in microbial engineering, namely, microbial consortium engineering, where the behavior of a multicultural microbial community rather than that of a single cell/species is delineated. Integrating the disciplines of microfluidics and microbial engineering opens up many new opportunities, for example in diagnostics, engineering of microbial motors, development of portable devices for genetics, high throughput characterization of genetic mutants, isolation and identification of rare/unculturable microbial species, single-cell analysis with high spatio-temporal resolution, and exploration of natural microbial communities.

Modifying the endogenous electron fluxes of Rhodobacter sphaeroides 2.4.1 for improved electricity generation.

The purple bacteria Rhodobacter sphaeroides serve as a promising biocatalyst in the photo-microbial fuel cell system (photo-MFC). This gram-negative species performs highly efficient anoxygenic photosynthesis that ensures an anaerobic environment in the anode compartment. Previous studies incorporating R. sphaeroides into photo-MFC were conducted using platinum as the anode electrode. In this study, we detected a steady current generation of R. sphaeroides in a bioelectrochemical system where glassy carbon was the working electrode and a typical growth medium was the electrolyte. The bioelectricity generation synchronized with the supplementation of reduced carbon source and showed immediate response to illumination, which strongly indicated the correlation between the observed current and the cytoplasmic quinone activity. Modifications of the endogenous electron flows mediated by quinone pool are shown to have significantly enhanced the bioelectricity generation. We anticipate that the findings in this study would advance future optimization of R. sphaeroides as an anode strain, as well as facilitate the study of bioenergetics in photosynthetic bacteria.

Tackling codon usage bias for heterologous expression in Rhodobacter sphaeroides by supplementation of rare tRNAs.

The photosynthetic Rhodobacter species are promising alternative expression hosts in bioproduction and biorefinery due to their unique metabolic capacities. With prominent inner membrane areas and efficient endogenous translocation machineries, they are especially attractive for membrane protein expression. However, codon usage bias could be a limitation in the engineering of Rhodobacter species and has seldom been investigated. In this study, we tackled the codon bias of Rhodobacter by functionally expressing 8 rare tRNAs of Rhodobacter sphaeroides with a multi-copy vector. The impact of tRNA supplementation was evaluated through monitoring expression levels of two heterologous proteins with different phylogenetic origins, a membrane subunit of the riboflavin transporter, RibU, from Lactobacillus acidophilus La-14 and a decaheme cytochrome, MtrA, from Shewanella oneidensis. Our results showed that the performances were closely related to medium composition and rare codon percentages of raw DNA sequences. Provision of rare tRNAs has increased RibU production by 7.7-folds and 2.86-fold in minimal medium and rich medium, respectively, while MtrA levels were increased by 1-fold in minimal medium. The present study confirms the presence of codon bias in R. sphaeroides and offers a facile tool for improving heterologous expression of rare-codon containing genes. We anticipate that this tRNA supplementation system can be further extended to other species of Rhodobacter, and thus will facilitate the engineering of purple bacteria for interesting applications in microbial technology.