Surface modification using a novel type I hydrophobin HGFI.

Surface wettability conversion with hydrophobins is important for its applications in biodevices. In this work, the application of a type I hydrophobin HGFI in surface wettability conversion on mica, glass, and poly(dimethylsiloxane) (PDMS) was investigated. X-ray photoelectron spectroscopy (XPS) and water-contact-angle (WCA) measurements indicated that HGFI modification could efficiently change the surface wettability. Data also showed that self-assembled HGFI had better stability than type II hydrophobin HFBI. Protein patterning and the following immunoassay illustrated that surface modification with HGFI should be a feasible strategy for biosensor device fabrication.

Self-assembled film of hydrophobins on gold surfaces and its application to electrochemical biosensing.

Hydrophobins are small fungal proteins which self-assemble on interfaces and significantly change the surface wettability. The self-assembled film of hydrophobin HFBI on a gold surface improved the surface hydrophilicity with water contact angle changing from 73.8+/-1.8 degrees to 45.3+/-1.4 degrees . A quartz crystal microbalance (QCM) analysis indicated that the HFBI coverage density on a gold surface was 588 ng cm(-2), and the self-assembled film remained stable under different pH values ranging from 1 to 13. A hydrophilic protein such as choline oxidase (ChOx) was then successfully immobilized on the HFBI modified gold surface. To evaluate the bioactivity of immobilized enzyme, an amperometric choline biosensor was constructed based on the Gold/HFBI/ChOx electrode, which produced as large as 4578.27 nA response current by 0.238 microg immobilized ChOx, when saturated by choline substrate. Comparing with our choline biosensors previously reported, the HFBI self-assembled film exhibited excellent capability to preserve the bioactivity of ChOx, hence a great potential in electrochemical biosensing is suggested.

Contribution of the conserved A16Leu to insulin foldability.

Contributions of the evolutionarily conserved A16Leu and B17Leu to insulin foldability were characterized by evaluating folding properties of single-chain insulin analogs. The results showed A16Leu had much more significant effects on the foldability of insulin than B17Leu.

Bioactive surface modification of mica and poly(dimethylsiloxane) with hydrophobins for protein immobilization.

Bioactive surfaces with appropriate hydrophilicity for protein immobilization can be achieved by hydrophobin II (HFBI) self-assembly on mica and polydimethylsiloxane (PDMS) surfaces. X-ray photoelectron spectroscopy and water contact angle measurements illustrated that the surface wettability can be changed from superhydrophobic (PDMS) or superhydrophilic (mica) to moderately hydrophilic, which is suitable for protein (chicken IgG) immobilization on both substrate surfaces. The results suggest that HFBI assembly, one kind of hydrophobin from Trichoderma reesei, may be a versatile and convenient method for the immobilization of biomolecules on diverse substrates, which may have potential applications in biosensors, immunoassays, and microfluidic networks.

Amperometric glucose biosensor based on self-assembly hydrophobin with high efficiency of enzyme utilization.

Hydrophobins are a family of natural self-assembling proteins with high biocompability, which are apt to form strong and ordered assembly onto many kinds of surfaces. These physical-chemical and biological properties make hydrophobins suitable for surface modification and biomolecule immobilization purposes. A class II hydrophobin HFBI was used as enzyme immobilization matrix on platinum electrode to construct amperometric glucose biosensor. Permeability of HFBI self-assembling film was optimized by selecting the proper HFBI concentration for electrode modification, in order to allow H(2)O(2) permeating while prevent interfering compounds accessing. HFBI self-assembly and glucose oxidase (GOx) immobilization was monitored by quartz crystal microbalance (QCM), and characterization of the modified electrode surface was obtained by scanning electron microscope (SEM). The resulting glucose biosensors showed rapid response time within 6s, limits of detection of 0.09 mM glucose (signal-to-noise ratio=3), wide linear range from 0.5 to 20mM, high sensitivity of 4.214 x 10(-3)AM(-1)cm(-2), also well selectivity, reproducibility and lifetime. The all-protein modified biosensor exhibited especially high efficiency of enzyme utilization, producing at most 712 microA responsive current for per unit activity of GOx. This work provided a promising new immobilization matrix with high biocompatibility and adequate electroactivity for further research in biosensing and other surface functionalizing.

[Identification of two new genes involved in swimming motility of Pseudomonas aeruginosa].

Pseudomonas aeruginosa, a typical opportunistic pathogen, has a single polar flagella which provides swimming motility and virulence. Twelve of insertional mutants with Mu transposition complexes into genomic DNA of Pseudomonas aeruginosa were screened on the changes of swimming motility. After gene cloning and DNA sequencing of those mutants, ten of them were illustrated that defective mutations on swimming motility were caused by insertions of Mu transposon into 10 of different functional genes which may involved in biosynthesis of flagella and gene regulations, but another two mutants were resulted from the insertion of two new genes (PA2950, PA5022) with function unknown. Observation of these two mutants with an integrity flagellum under electron microscope showed that two new genes were not involved in construction of flagella, but probably in energy metabolism and gene regulation of swimming motility.

[Study on genes involved in biosynthesis and regulation of pigments in Pseudomonas aeruginosa].

Mu transposition recombination technique was firstly used as a mutagenesis tool to explore a cluster of genes involved in biosynthesis and regulation of pigments in P. aeruginosa. Eight pigment mutants were screened and identified. Gene cloning and sequencing of the region flanking the insertion revealed that the genes hmgA, ptsP, sucC, phzS, phzF1 were disrupted with mini-Mu respectively. Among them, gene hmgA is involved in the degradation of tyrosine, others affect the metabolism of pyocyanin which is the most important pigment of P. aeruginosa. Both gene phzS and gene phzF1 have been experimentally demonstrated to participate in pyocyanin synthesis. Gene ptsP belongs to a phosphotransferase system and gene sucC encodes succinyl-CoA synthetase beta chain. This is the first report that gene ptsP and sucC may be involved in the regulation of the biosynthesis of pyocyanin.

[The study of optimal conditions of electroporation in Pseudomonas aeruginosa].

A P. aeruginosa strain PA68 isolated from the sputum of a patient suffering from bronchiectasis was used as the recipient strain. Optimum conditions including growth stage of the strain, electroshock voltage, concentration and preservation of competent cell were defined for the electroporation of PA68 with plasmid pSMC28. It was showed that the highest transformation efficiency was up to 1.68 x 10(3) CFU/microgram DNA under the optimum conditions in which the competent cells were collected at logarithmic growth phase (OD(540) = 0.7-0.8) and concentrated to about 10(11) cells/ml, the mixture of the competent cells and plasmid pSMC28 was eletroporated at 2.6 kV. With this optimal condition, Mu transponson complexes have been successfully transformed into P. aeruginosa strain PA68 and the obtained efficiency was 2.47 x 10(4) CFU/microgram DNA. This is the first time to electroporate Mu transposon complexes into Pseudomonas spp. The artificial Mu transposons could integrate into bacterial genomes at a single site randomly. Then the phenotype change was the result of the gene inactivation caused by Mu transposon insertion. That will be very helpful for the study of genomic function of Pseudomonas spp.

[The action of S1 nuclease and a cloning strategy for microcircular DNAs].

S1 nuclease (from Aspergillus oryzae) is a specific enzyme to degrade single stranded DNA or RNA molecules. It has been reported to be able to convert superhelical circular DNA molecules into open circle or linear forms under certain conditions, but this function has not been well explored. In order to use the action of S1 nuclease to linearize circular DNA and develop a novel way of cloning microcircular DNAs, the pUC19 was used to investigate the relationship between the linearization efficiency of S1 nuclease and the amount of enzyme used. By this way the optimal conditions for linearization of circular DNAs by S1 nuclease would be determined. 0.3u to 17u S1 nuclease per 100ng pUC19 DNA was added into a 25 microL system, respectively, to perform the reaction. The effectiveness of enzyme digestion was realized by electrophoresis in a 1.2% agarose gel. The results showed that along with the increase in enzyme amount from 0.3u to 17u a gradual decrease in the superhelical form, a gradual increase in the linear form and then in the circular form was obvious. The conversion from superhelical form to linear and circular form was directly related to the enzyme amount used. A higher proportion of linear DNA molecules was achieved by using 5 to 17u S1 nuclease per 100ng DNA. Besides, electrophoretic mobility of the S1 nuclease-linearized pUC19 was the same as that of the linear form produced by restriction enzyme digestion. According to the result of phiX174 digested by S1 nuclease it has been proposed that the enzyme cleaves first randomly on one site of one strand, thus converting the superhelical molecules into open circle form, and then on the same site of the complementary strand to produce the linear form. Therefore, the S1 nuclease-linearized DNA molecules are intact in the sense of their length and can be used for cloning. The plasmid-like DNA pC3 from cucumber mitochondria is a double stranded circular DNA molecule with about 550bp and the smallest known plasmid-like DNA in eukaryotic mitochondria. Many attempts have been made to linearize the molecule by using restriction enzymes but failed. Therefore, S1 nuclease was used to linearize pC3 based on the results obtained with pUC19. The linearized pC3 DNA molecules formed a very sharp band in a 2.5% agarose gel after electrophoresis. They were then recovered from the gel, added an "A" tail and ligated with T-vector. After transformation into E. coli JM109 cells, the positive clones were, screened by the blue-white selection. The insert was then cut using restriction enzymes EcoRI and Pst I. The result of electrophoresis shows that the electrophoretic mobility of the insert is just the same as that predicted. A 32 P-labled probe was synthesized using pC3 as the template and Southern blot analysis was carried out. The result shows that the inserted DNA is hybridized to the probe, which indicates that the cloned DNA fragment is from pC3. The sequence information of the insert shows that the plasmid-like DNA pC3 was 537bp in length. The nucleotide sequence was deposited in the GenBank (the accession number is AF522195).