Search

Biocontainment is a key methodology to reduce environmental risk through the deliberate release of genetically modified microorganisms. Previously, we developed a phosphite (HPO 3 2- )-dependent biocontainment strategy, by expressing a phosphite-specific transporter HtxBCDE and phosphite dehydrogenase in bacteria devoid of their indigenous phosphate (HPO 4 2- ) transporters. This strategy did not allow Escherichia coli to generate escape mutants (EMs) in growth media containing phosphate as a phosphorus source using an assay with a detection limit of 1.9 × 10 -13 . In this study, we found that the coexistence of a high dose of phosphate (>0.5 mM) with phosphite in the growth medium allows the phosphite-dependent E. coli strain to generate EMs at a frequency of approximately 5.4 × 10 -10 . In all EMs, the mutation was a single amino acid substitution of phenylalanine to cysteine or serine at position 210 of HtxC, the transmembrane domain protein of the phosphorus compound transporter HtxBCDE. Replacement of the HtxC F210 residue with the other 17 amino acids revealed that HtxC F210 is crucial in determining substrate specificity of HtxBCDE. Based on the finding of the role of HtxC F210 as a "gatekeeper" residue for this transporter, we demonstrate that the replacement of HtxC F210 with amino acids resulting from codons that require two simultaneous point mutations to generate phosphate permissive HtxC mutants can reduce the rate of EM generation to an undetectable level. These findings also provide novel insights into the functional classification of HtxBCDE as a noncanonical ATP-binding cassette transporter in which the transmembrane domain protein participates in substrate recognition.

Recent progress in genetic engineering and synthetic biology have greatly expanded the production capabilities of cyanobacteria, but concerns regarding biosafety issues and the risk of contamination of cultures in outdoor culture conditions remain to be resolved. With this dual goal in mind, we applied the recently established biological containment strategy based on phosphite (H 3 PO 3 , Pt) dependency to the model cyanobacterium Synechococcus elongatus PCC 7942 ( Syn 7942). Pt assimilation capability was conferred on Syn 7942 by the introduction of Pt dehydrogenase (PtxD) and hypophosphite transporter (HtxBCDE) genes that allow the uptake of Pt, but not phosphate (H 3 PO 4 , Pi). We then identified and disrupted the two indigenous Pi transporters, pst (Synpcc7942_2441 to 2445) and pit (Synpcc7942_0184). The resultant strain failed to grow on any media containing various types of P compounds other than Pt. The strain did not yield any escape mutants for at least 28 days with a detection limit of 3.6 × 10 -11 per colony forming unit, and rapidly lost viability in the absence of Pt. Moreover, growth competition of the Pt-dependent strain with wild-type cyanobacteria revealed that the Pt-dependent strain could dominate in cultures containing Pt as the sole P source. Because Pt is rarely available in aquatic environments this strategy can contribute to both biosafety and contamination management of genetically engineered cyanobacteria.

Water-soluble cyclodextrin (CyD) complexed with porphyrin derivatives with different substituents in the meso -positions showed different photodynamic activities toward cancer cells under illumination at wavelengths over 600 nm, the most suitable wavelengths for photodynamic therapy (PDT). In particular, aniline- and phenol-substituted derivatives had high photodynamic activity because of the efficient intracellular uptake of the complexes by tumor cells. These complexes showed greater photodynamic activity than photofrin, currently the main drug in clinical use as a photosensitizer. These results represent a significant step toward the optimization of porphyrin derivatives as photosensitizers.

A new homogeneous insulin assay requiring no chemical modification of an insulin recognition domain, which can be applied to continuous monitoring of the time-dependent cellular response in vitro, was developed. The carboxy-terminal α-chain (αCT) segment and first leucine-rich-repeat (L1) domain in the primary binding site on the insulin receptor were genetically fused with a bioluminescent protein (Nanoluc, Nluc) and a fluorescent protein (yellow fluorescent protein, YPet) to produce the insulin-sensing probe proteins Nluc-αCT and L1-YPet. The BRET signal was observed on simple mixing of insulin with these protein probes, in a so-called homogeneous assay. The BRET signal was proportional to the insulin concentration, and the lower detection limit was 0.8 μM. Time-dependent insulin secretion from drug-stimulated MIN6 cells was also successfully monitored continuously with the probe proteins. This BRET-based homogeneous insulin assay method is thus expected to be applicable to drug development by high-throughput screening.

Protein immune detection requires secondary antibodies which must be carefully selected in order to avoid interspecies cross-reactivity, and is therefore restricted by the limited availability of primary/secondary antibody pairs. Here we present a versatile DNA-based protein detection system using a universal adapter to interface between IgG antibodies and DNA-modified reporter molecules. As a demonstration of this capability, we successfully used DNA nano-barcodes, quantum dots, and horseradish peroxidase enzyme to detect multiple proteins using our DNA-based labeling system. Our system not only eliminates secondary antibodies but also serves as a novel method platform for protein detection with modularity, high capacity, and multiplexed capability.

It is currently not clear how to quantifiably relate pore-scale observations of colloid transportto larger scales, so,we proposed a geometric theory showing that pore-scale-derived rate constants may be appropriate to model a larger scale system. This study considered three different types of colloids: latex microspheres, Escherichia coli, and microspheres made of poly lactic acid (PLA). Colloid attachment and detachment rate constants were calculated using digital microscope images, taken in rapid (1 s) sequences, from which rates of attaching and detaching colloids were readily observed. Average rate constants from >1000 images per colloid-type were used to model Darcy-scale colloid transport breakthrough curves. The modeled and observed breakthrough curves agreed well for all three types of colloids. However, for latex and PLA microspheres, the model systematically under predicted the breakthrough curves' rising limb, which may indicate that the rate "constants" are actually dependent on the amount of attached colloids. Insights into these sorts of complexities are best addressed by research that considers both pore-scale phenomena and larger-scale transport responses.

It is widely recognized that stimuli-responsive nanostructures play a promising role in nanodevices for medical treatments and experimental tools. We have designed and constructed a basic structure which controls the distance between two termini domains through temperature reversibility. Our structure, shaped like a bouquet, is composed of two proteins, alpha-helix and elastin-like protein (ELP). Alpha-helices align and bundle the ELP while ELP twists and forms a fiber-like structure at warm temperatures. This ELP conformational change alters the distance between the structure termini at the site opposite the alpha-helix. We connected enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) at the structure's two termini to evaluate the distance using fluorescence resonance energy transfer (FRET) efficiency. These proteins spontaneously formed a complex which decreased the distance between the two fluorescent proteins located at its termini, at physiologically relevant temperatures. This change was repeated with complete reversibility (n = 5).

Intermolecular enzyme complementation assay is a useful method for detecting protein-protein interactions. Specifically, bioluminescent signals produced from reconstructed split luciferase fragments are powerful tools for in vivo analysis because the bioluminescent signals have been visualized both in cultured cells and living animals. However, they are limited for detection and evaluation of biological events relevant to intermolecular protein-protein interactions. In this study, we constructed an intramolecular luciferase complementation probe for detecting target biomolecules other than protein-protein interactions. It consists of peptide-inserted firefly luciferase (PI-FLuc) containing a short peptide between internally divided firefly luciferase. The inserted short peptide triggers FLuc complementation or discomplementation and subsequent reactivation or inactivation of FLuc activity through its induced fit conformational changes. We chose RNA binding arginine rich motif (ARM) peptides, Rev and/or Tat, for model peptide insertion, and expressed constructed PI-FLuc probe variants using a wheat germ cell-free protein synthesis system. They showed FLuc activity changes, reactivation, or inactivation after binding to their specific RNA targets. Furthermore, to expand the versatility of the PI-FLuc RNA detection system, we designed split-RNA probes built to reform the ARM peptide binding site in the presence of arbitrarily selected target-RNA. As a result, the target RNA was homogeneously detected by FLuc luminescent signals mediated by a cooperative function of the PI-FLuc and split-RNA probe sets.

Detection of specific nucleic acids is important to understand cellular mechanisms and functions of gene regulation. Here, we demonstrated a novel method to detect specific nucleic acids using recombinant protein and oligonucleotides. A recombinant protein YRGnC-11ad, which has a Rev-peptide between enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) was constructed and expressed in HeLa cells. Rev-peptide, which corresponds to amino acids 34-50 of the HIV-1 Rev protein, indicates disordered structure in solution but forms alpha-helical and elongated conformation upon binding to Rev response element RNA (RRE-RNA) and Rev-aptamer, respectively. We confirmed that YRGnC-11ad could specifically bind to RRE-RNA and Rev-aptamer in cell lysate, and fluorescent resonance energy transfer (FRET) signal was changed upon binding following the conformational change of Rev-peptide. To utilize this FRET signal change toward the detection of specific nucleic acids, we split the RRE-RNA sequence and connected to the complementary oligonucleotide for target nucleic acids. When each two oligonucleotides hybridized to an adjacent region of target nucleic acids correctly, a Rev-peptide binding site was reformed on the hybridized complex. And we could confirm that YRGnC-11ad recombinant protein indicated FRET increase upon binding to the hybridized complex in cell lysate. These results suggest that the recombinant protein probe is available for specific nucleic acid detection.