[Show abstract][Hide abstract] ABSTRACT: Nanoemulsions are useful as drug carriers. Conjugating targeting molecules to nanoemulsions would expand their application to the cell-specific delivery of drugs. Click reactions, such as the Cu (I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reaction, are a convenient tool for such functionalization as the conjugation of label or targeting molecules. In this study, we sought to synthesize siloxane-based amphiphilic polymers containing alkyne moieties to form nanoemulsions capable of conjugating azide-functionalized labels by the CuAAC reaction. The siloxane-based homopolymer (P1) and copolymer with polydimethylsiloxane (P2) containing chloropropyl groups were reacted with an imidazole derivative that had an ethynyl group at the 1-position to generate PIm1 and PIm2, respectively. PIm1 and PIm2 exhibited good solubility in polar solvents such as methanol, dimethylformamide, dimethyl sulfoxide and H 2O. Further, PIm2 exhibited solubility in chloroform and dichloromethane. Azide-functionalized fluorescent dyes and green fluorescent proteins were conjugated with PIm1 and PIm2 by the CuAAC reaction. The labeled polymer emulsified soybean oil in water to form emulsions. The average emulsion size was 150 nm. The fluorescent labels on the emulsions were successfully observed with a fluorescent microscope. Conjugation of PIm1 and PIm2 with targeting molecules by the click reaction would be useful for cell-specific drug delivery.
[Show abstract][Hide abstract] ABSTRACT: Serine-type phage integrases catalyze unidirectional site-specific recombination between the attachment sites, attP and attB, in the phage and host bacterial genomes, respectively; these integrases and DNA target sites function efficiently when transferred into heterologous cells. We previously developed an in vivo site-specific genomic integration system based on actinophage TG1 integrase that introduces ∼2-kbp DNA into an att site inserted into a heterologous Escherichia coli genome. Here, we analyzed the TG1 integrase-mediated integrations of att site-containing ∼10-kbp DNA into the corresponding att site pre-inserted into various genomic locations; moreover, we developed a system that introduces ∼10-kbp DNA into the genome with an efficiency of ∼104 transformants/μg DNA. Integrations of attB-containing DNA into an attP-containing genome were more efficient than integrations of attP-containing DNA into an attB-containing genome, and integrations targeting attP inserted near the replication origin, oriC, and the E. coli “centromere” analogue, migS, were more efficient than those targeting attP within other regions of the genome. Because the genomic region proximal to the oriC and migS sites is located at the extreme poles of the cell during chromosomal segregation, the oriC–migS region may be more exposed to the cytosol than are other regions of the E. coli chromosome. Thus, accessibility of pre-inserted attP to attB-containing incoming DNA may be crucial for the integration efficiency by serine-type integrases in heterologous cells. These results may be beneficial to the development of serine-type integrases-based genomic integration systems for various bacterial species.
No preview · Article · Oct 2012 · Applied Microbiology and Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Poly(tetramethylsilarylenesiloxane) derivatives having diphenylfluorene (P1) or diphenyldibenzosilole (P2) moieties were prepared via polycondensation of the corresponding disilanol monomers, that is, 2,7-bis(dimethylhydroxysilyl)-9,9-diphenylfluorene (M1) and 2,7-bis(dimethylhydroxysilyl)-9,9-diphenyldibenzosilole (M2), respectively. P1 and P2 exhibited good solubility in common organic solvents. The glass transition temperatures (Tgs) of P1 and P2 were determined by differential scanning calorimetry to be 125 and 119 °C, respectively. The melting temperature (Tm) of P1 was observed at 276 °C; however, the Tm of P2 was not observed, indicating that the introduction of a dibenzosilole moiety decreased the crystallization tendency. The temperatures at 5% weight loss (Td5s) of P1 and P2 were 539 and 520 °C, respectively, suggesting good thermostability of P1 and P2. Bathochromic and hyperchromic effects were observed in the absorption and fluorescence spectra by introducing a dimethylsilyl substituent onto diphenylfluorene and diphenyldibenzosilole skeletons. The replacement of diphenylfluorene by the corresponding diphenyldibenzosilole also led to bathochromic shifts. The fluorescence quantum yield (ΦF) of P1 was lower than that of M1, probably because of the formation of aggregates; however, the ΦF of P2 was higher than that of M2, indicating a decrease in the tendency toward aggregation using a dibenzosilole skeleton.
[Show abstract][Hide abstract] ABSTRACT: Phage integrases are enzymes that catalyze unidirectional site-specific recombination between the attachment sites of phage and host bacteria, attP and attB, respectively. We recently developed an in vivo intra-molecular site-specific recombination system based on actinophage TG1 serine-type integrase that efficiently acts between attP and attB on a single plasmid DNA in heterologous Escherichia coli cells. Here, we developed an in vivo inter-molecular site-specific recombination system that efficiently acted between the att site on exogenous non-replicative plasmid DNA and the corresponding att site on endogenous plasmid or genomic DNA in E. coli cells, and the recombination efficiencies increased by a factor of ~10(1-3) in cells expressing TG1 integrase over those without. Moreover, integration of attB-containing incoming plasmid DNA into attP-inserted E. coli genome was more efficient than that of the reverse substrate configuration. Together with our previous result that purified TG1 integrase functions efficiently without auxiliary host factors in vitro, these in vivo results indicate that TG1 integrase may be able to introduce attB-containing circular DNAs efficiently into attP-inserted genomes of many bacterial species in a site-specific and unidirectional manner. This system thus may be beneficial to genome engineering for a wide variety of bacterial species.
No preview · Article · Nov 2010 · Applied Microbiology and Biotechnology