alpha 2,3-Sialyltransferase from Pasteurella multocida (PmST1) is an enzyme that transfers a sialyl group of donor substrates to an acceptor substrate called N-acetyl-D-lactosamine (LacNAc). In this study PmST1 was expressed on the outer membrane of wildtype Escherichia coli (BL21) with lipopolysaccharide (LPS) and ClearColi with no LPS, and then the enzyme activity and expression level of PmST1 were compared. As the first step, the expression levels of PmST1 on the outer membranes of wildtype E. coli (BL21) and ClearColi were compared according to the IPTG induction time, and the absolute amount of surface-displayed PmST1 was calculated using densitometry of SDS-PAGE. As the next step, the influence of LPS on the PmST1 activity was estimated by analyzing Michaelis-Menten plot. The enzyme activity of PmST1 was analyzed by measuring the concentration of CMP, which was a by-product after the transfer of the sialyl group of donor compounds to the acceptor compounds. From a Michaelis-Menten plot, the enzyme activity of the surface-displayed PmST1 and the maximum rate (V-max) of ClearColi were higher than those of wildtype E. coli (BL21). However, the K M value, which represented the concentration of substrate to reach half the maximum rate (V-ma(x)), was similar for both enzymes. These results represented such a difference in enzyme activity was occurred from the interference of LPS on the mass transport of the donor and acceptor to PmST1 for the sialyl group transfer.

The autoimmune diseases systemic lupus erythematosus (SLE) and Sjogren's syndrome (SS) are diagnosed by detection of autoantibodies against Ro and La protein autoantigens, respectively. In this work, the diagnosis of autoimmune diseases SLE and SS was demonstrated using thermophoresis of Escherichia coli with the autodisplayed autoantigens. Ro and La protein autoantigens were autodisplayed by constitutive expression together with a fluorescent protein called tdTomato in the cytosol. The binding affinity of the autodisplayed autoantigens was tested against positive and control sera by using FACS as a reference method. The factors influencing interactions between E. coli with autodisplayed autoantigens and autoantibodies in sera during thermophoresis were analyzed by measurement of cell surface charge and size before and after interaction. Finally, the thermophoretic diagnosis of autoimmune diseases SLE and SS was demonstrated using sera from patients afflicted with the respective diseases by estimating sensitivity and selectivity from ROC plots.

Harvesting photosynthetic electrons (PEs) from plant or algal cells can be a highly efficient and environmentally friendly way of generating renewable energy. Recent work on nanoelectrode insertion into algal cells has demonstrated the possibility to directly extract PEs from living algal cells with high efficiencies. However, the instability of the inserted cells limits the practicality of this technology. Here, the impact of nanoelectrode insertion on intracellular extraction of PEs is characterized with the goal of stabilizing algal cells after nanoelectrode insertion. Using nanoelectrodes <500 nm in diameter, algal cells remained stable for over one week after insertion and continued to provide PEs through direct extraction by the inserted nanoelectrodes. After nanoelectrode insertion, a photosynthetic current density of 6 mA·cm−2, which is several fold higher than the current densities attained using approaches based on isolated thylakoid membranes or photosystem I complexes, was observed in the dark and during illumination at various light intensities.

Plant cells produce photosynthetic electrons (PEs) by splitting water during photosynthesis. In the beginning of photosynthesis, antenna complex embedded in thylakoid membrane absorbs photon energy and converts them into excited electrons with high quantum efficiency. These excited electrons, known as PEs, are transferred through electron acceptors in order to grow cell or store surplus energy as a form of carbonhydrates. However, since the energy state of the PEs is lowered once they are stored as organic matter, many researchers have tried to extract PEs from photosynthetic electron transfer chain before their conversion into organic matter. Such previous works include collection of PEs by electrochemical oxidizing through mediator as an electron interceptor and use of electrical linker materials such as CNTs, electrically-conducting polymer matrix, or conducting linker to working electrodes between photosynthetic components and the surface of electrodes. Although these works demonstrated the feasibility of harvesting PEs in large scale, there are still issues including efficiency decrease from mediator use and limited stability or requirement of additional electron donors of isolated photosynthetic extracts. In our previous study, we demonstrated direct extraction of PEs from living algal cell, chlamydomonas reinhardtii, by inserting nanoelectrode (NE) into cell membrane. However, in spite of highly efficient extraction of PEs, the maximum harvesting period of PEs was shorter than 1 hour. In this study, we developed a cantilever type of NE system in order to find the optimal shape and size of NE for extraction of PEs for a longer period of time. Fabrication of cantilever NE system started by milling the end of commercial AFM cantilever tip by focused ion beam (FIB). Then, Au was deposited on the cantilever by sputtering as a working electrode. Si 3 N 4 was coated on top of the Au-coated NE to minimize noise signals. Then, additional FIB milling was performed to expose the Au layer at the tip of the NE for localized PE signal measurement. With this cantilever NE, we firstly investigated a relationship between NE size and the viability of inserted cells. It was confirmed that algal cells remained stable for up to 7 days when NE diameters smaller than 500 nm were used. Through continuous observation of inserted algal cells, their viability was ascertained by cell proliferation. After high stability of NE inserted cells was confirmed, direct extraction of PEs was attempted without any mediator. Light-triggered currents from the cells inserted by NEs were observed and the photosynthetic origin of the currents was confirmed by application of DCMU and the subsequent disappearance of the light-responsive currents. Since exposure to highly intense and continuous illumination can damage the pigments of the photosystems, the effect of illumination condition on the PE extraction was investigated. Up to the light intensity of 132 μmol photons m -2 s -1 , no photobleaching was observed from the NE-inserted cells and the stronger light intensity produced the higher photosynthetic currents. When NE-inserted cells were incubated in a dark except during illumination for photosynthetic current harvesting, about 150 fA of photosynthetic currents were measured over 7 days with 9 μmol photons m -2 s -1 of light intensity at 400 mV against Ag/AgCl electrode. As another comparison, when NE-inserted cells were exposed to continuous illumination with 16.5 μmol photons m -2 s -1 for 60 hours, more than twice amount of PEs was measured compared to dark incubated cells. Finally, NE-inserted cells were exposed to continuous illumination and th e photosynthetic currents were monitored simultaneously. It was shown that PEs were extracted steadily during several hours of illumination. These results indicated that PEs could be extracted for an extended period of time under various illumination conditions. In this work, the maximum photosynthetic currents measured were 2.5 pA after continuous illumination on a single NE-inserted cell.

The objective of this study was to present an immunoassay for the diagnosis of Sjögren's syndrome based on the autodisplayed La/SSB protein on the outer membrane of intact E. coli (strain UT-5600) and LPS-free E. coli (ClearColi™). As the first step, an autodisplay vector (pCK002) was transfected into intact E. coli and LPS-free E. coli for comparison of efficiency of autdisplay of La/SSB. The maximal level of La/SSB expression was estimated to be similar for LPS-free E. coli and intact E. coli at different optimal induction periods. Intact E. coli was found to grow twofold faster than LPS-free E. coli, and the maximal level of expression for LPS-free E. coli was obtained with a longer induction period. When the zeta potential was measured, both intact E. coli and LPS-free E. coli showed negative values, and the autodisplay of negatively charged La/SSB protein (pI<7) on the outer membrane of intact E. coli and LPS-free E. coli resulted in a slight change in zeta potential values. E. coli with autodisplayed La/SSB protein was used for an immunoassay of anti-La/SSB antibodies for the diagnosis of Sjögren's syndrome. The surface of E. coli with the autodisplayed antigen was modified with rabbit serum and papain to prevent false positive signals because of nonspecific binding of unrelated antibodies from human serum. LPS-free E. coli with autodisplayed La/SSB protein yielded sensitivity and selectivity of 81.6% and 78.6%, respectively. The Bland-Altman test showed that the immunoassays based on LPS-free E. coli and intact E. coli with autodisplayed La/SSB protein were statistically equivalent to a clinical immunoassay for detection of anti-La/SSB antibodies (confidence coefficient 95%).

The autodisplay technology has been applied for expression of a desired protein on the outer membrane (OM) of Escherichia coli. In this work, the OM fractions of E. coli with two autodisplayed proteins were separately prepared and mixed to demonstrate the feasibility of control over the ratio of two autodisplayed proteins. As the first model, Z-domain and streptavidin were autodisplayed, and their activities were tested by means of the combined OM layer in a 96-well microplate and a surface plasmon resonance (SPR) biosensor. As the second model, lipase and foldase were autodisplayed which required an interaction between two proteins to obtain the activity of lipase. The OM fractions of E. coli with an autodisplayed lipase and foldase were separately prepared and mixed to demonstrate the feasibility of control over the ratio of two autodisplayed proteins when the interaction of two proteins is required within the same OM layer for the activity of the lipase.

The Z-domain has the potential to control the orientation of immobilized antibodies because of its binding affinity to the Fc regions of antibodies (IgGs). In this work, Z-domains were autodisplayed on the outer membrane (OM) of Escherichia coli. OM particles were isolated and coated onto microbeads with positive, neutral, or negative surface charges. Other conditions such as incubation time and initial OM concentration were also optimized for the OM coating to obtain maximum antibody-binding. Using three kinds of model proteins with different isoelectric points (pI), streptavidin (pI = 5, negative charge at pH 7), horseradish peroxidase (pI = 7, neutral charge at pH 7), and avidin (pI = 10, positive charge at pH 7), protein immobilization onto the microbeads was carried out through physical adsorption and electrostatic interactions. Using fluorescently labeled antibodies and fluorescence-activated cell sorting, it was determined that the neutral and the positively charged microbeads effectively bound antibodies while minimizing non-specific protein binding. The OM-coated microbeads with autodisplayed Z-domains were applied to C-reactive protein immunoassay. This immunoassay achieved 5-fold improved sensitivity compared to conventional immunoassay based on physical adsorption of antibodies at the cutoff concentration of medical diagnosis of inflammatory diseases (1000 ng/ml) and cardiovascular diseases (200 ng/ml). (C) 2015 Elsevier B.V. All rights reserved.

Escherichia coli cells with autodisplayed Z-domains have been used for immunoassays of specific target analytes. In this study, a magnetite suspension was used for the washing step in immunoassays of E. coli cells with autodisplayed Z-domains. This approach enhanced the washing conditions for these immunoassays by determining (1) the optimal concentration of the magnetite suspension, (2) the capacity of the magnetite suspension-based washing method to recover E. coli cells, and (3) the level at which the activity of autodisplayed Z-domains is maintained. In immunoassays of C-reactive protein (CRP), the immunoassay incorporating the magnetite suspension-based washing method showed a sensitivity and limit of detection considerably higher than those of the conventional centrifugation-based washing method. The results indicated that immunoassays incorporating the magnetite suspension-based washing method are effective for medical diagnoses based on CRP assay.

An electrochemically active nanowire system is fabricated using wet‐tapped nanosphere lithography and a single photolithography step. The patterned nanowire/nanoelectrode is inserted into live algal cells, enabling the potential harvesting of photosynthetic electrons from multiple cells simultaneously. Light‐dependent extraction of electrons from cells is observed; these electrons are derived from the photosynthetic electron transport chain based on a light intensity‐dependence of the reaction coupled with the finding that electron extraction is inhibited in the presence of DCMU (3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea), a reagent that specifically blocks electron flow out of photosystem II. Insertion of nanoelectrodes into multiple algal cells is achieved, and sequential insertion of cells with the nanoelectrode, followed by subsequent removal of the electrode, yields a corresponding increase and then decrease in light‐driven currents. Controlling the intensity of the illumination avoids nearly all photodamage and enables direct extraction of more photosynthetic electrons from multiple cells in parallel, which is sustained for an extended period of time.

In this work, adrenodoxin (Adx) was expressed on the outer membrane of E. coli by autodisplay and then the iron–sulfur cluster was incorporated into apo-Adx by an anaerobic reconstitution process. For the determination of the redox potentials of the iron–sulfur clusters of the autodisplayed Adx, E. coli cells with autodisplayed Adx were immobilized on a gold electrode modified with a self-assembled monolayer of mercaptoundecanoic acid (MUA). From the repeated cyclic voltammetry (CV) analysis, the E. coli (10 mM HEPES buffer, pH 7.0) with autodisplayed Adx showed significant changes in shape with an oxidation peak at + 0.4 V (vs. Ag/AgCl) and a reduction peak at − 0.3 V (vs. Ag/AgCl) after the reconstitution process for the incorporation of the iron–sulfur cluster. From the repeated CV analysis in the reduction and oxidation potential ranges, the iron–sulfur clusters of the autodisplayed Adx were observed to undergo reversible redox reactions via direct electron transfer to the MUA-modified gold electrode.

"Autodisplay technology" is an expression technique used to display the various recombinant proteins on the outer membrane (OM) of Escherichia coli. The resulting autodisplayed Z-domain has been used to improve the sensitivity of immunoassays. In this work, a facile isolation method of the OM fraction of E. coli with autodisplayed Z-domains was presented using (1) an enzyme reaction for the hydrolysis of the peptidoglycan layer and (2) short centrifugation steps. The purity of the isolated OM fraction was analyzed. For the estimation of contamination with bacterial proteins from other parts of E. coli, Western blots of marker proteins for the OM (OmpA), periplasm (β-lactamase), inner membrane (SecA), and cytoplasm (β-galactosidase) were performed. Additionally, assays of marker components or enzymes from each part of E. coli were carried out including the OM (KDO), inner membrane (NADH oxidase), periplasm (β-lactamase), and cytoplasm (β-galactosidase). The yield of OM isolation using this new method was determined to be 80% of the total OM amount, with less than 1% being contaminants from other parts of E. coli.

In this work, two proteins, Z-domains and bovine casein, were autodisplayed on the outer membrane of the same Escherichia coli cells by co-transformation of two different autodisplay vectors. On the basis of SDS-PAGE densitometry, Z-domains and bovine casein were expressed at 3.12×105 and 1.55×105 proteins/E. coli cell, respectively. The co-autodisplayed Z-domains had antibody-binding activity and the bovine casein had adhesive properties. E. coli with co-autodisplayed proteins were analyzed by fluorescence assisted cell sorting (FACS). E. coli with co-autodisplayed Z-domains and bovine casein aggregated due to hydrophobic interaction. For application to immunoassays, the Z-domain activity was estimated after (1) immobilizing the E. coli and (2) forming an OM layer. E. coli with co-autodisplayed two proteins that were immobilized on a polystyrene microplate had the same antibody-binding activity as did E. coli with autodisplayed Z-domains only. The OM layer from the co-transformed E. coli had Z-domains and bovine casein expressed at a 1:2 ratio from antibody-binding activity measurements.

Fluorescence-activated cell sorter (FACS)-based immunoassays using E. coli cells with autodisplayed Z-domains were performed to (1) improve the sensitivity of the immunoassay through orientation control of antibodies with autodisplayed Z-domains and (2) minimize the required amount of analyte by using FACS to measure the fluorescent signal from individual E. coli cells. The expression (autodisplay) of Z-domains on the outer membrane of E. coli was confirmed by fluorescence image analysis, and the homogeneous distribution of autodisplayed Z-domains was presented by SEM image analysis after treatment with antibodies labeled with gold nanoparticles (diameter of 15 nm). As FACS measures the fluorescent signal from individual E. coli cells, the optimal FACS parameters for the effective detection of fluorescently labeled E. coli cells were determined, and the minimum amount of analyte required for the E. coli cell-based immunoassay was identified using two model immunoassay configurations. Finally, the medical diagnosis of heart infarction with a biomarker called troponin-I using the E. coli cell-based immunoassay with FACS analysis was demonstrated.

A microarray-based immunoassay for the detection of autoantibodies against Ro protein was developed using Escherichia coli with autodisplayed Ro proteins (Ro(+)-E. coli). Patient serum usually contains various antibodies against the outer membrane components of E. coli as well as autoantibodies against the Ro protein. Therefore, the conventional immunoassay based on Ro(+)-E. coli requires both wild type E. coli (blank test) and Ro(+)-E. coli, and both strains of E. coli must be prepared in situ for each individual test serum. In this study, we tested the feasibility of using several types of animal sera as a replacement for individual human sera. An immunoassay without the blank test was developed using Ro(+)-E. coli by (1) blocking with rabbit serum, and (2) cleaving the Fc region from antibodies using papain. Modified E. coli with autodisplayed Ro protein was immobilized to a surface-modified microplate and the applicability of the immunoassay without the blank test was demonstrated using sera from patients with systemic lupus erythematosus (SLE). Using this approach, a microarray-based fluorescence immunoassay with immobilized Ro(+)-E. coli was able to detect anti-Ro autoantibodies in SLE patient sera with high specificity and selectivity and improved efficiency.

Escherichia coli cells with autodisplayed Z-domains could increase the sensitivity of immunoassays by immobilizing antibodies in a controlled orientation. In the work presented here, E. coli cells with autodisplayed Z-domains were immobilized to magnetic beads for subsequent immunoassay. In comparing conventional immunoassay using the E. coli cells with autodisplayed Z-domains, the magnetic-bead-based immunoassay improved immunoassay efficiency by minimizing the loss of E. coli cells during repeated centrifugation steps during washing. For the immobilization of E. coli cells to magnetic beads, the magnetic beads were modified with poly-l-lysine to bind to negatively charged E. coli cells. During the surface modification process, physical parameters such as the surface charge and size of the magnetic beads were analyzed to confirm the formation of E. coli-magnetic bead complexes. To test the feasibility of the magnetic-bead-based immunoassay, horseradish peroxidase (HRP) was used as a model analyte, and a biomarker for inflammatory diseases, C-reactive protein (CRP), was used for a demonstration of an application in medical diagnosis.

Recently, we reported a highly sensitive immunoassay using Escherichia coli cells with autodisplayed Z-domains. In this work, E. coli cells with autodisplayed Z-domains were applied to the flow-cytometry-based simultaneous detection of multiple analytes. The E. coli cells were doubly transfected to express a fluorescent protein (tdTomato) in the cytosol and the autodisplayed Z-domains on the outer membrane. By using E. coli cells with only the autodisplayed Z-domains, immunoassay of multiple analytes could be performed simultaneously on the same sample. Flow cytometry can be used to identify the immunoassay type by simultaneously detecting the fluorescence signal from the cytosol (tdTomato) and the fluorescence from the outer membrane, enabling the quantification of bound analytes after treatment with additional fluorescently labeled antibodies. To demonstrate the immunoassay of multiple analytes by using flow cytometry, human hepatitis B virus surface antigen (HBsAg) and C-reactive protein (CRP), a broad spectrum inflammation marker, were used as model analytes.