Earticle

We report a novel method to fabricate poly(vinyl alcohol) (PVA) hydrogel nanopatterns for constructing the nanoarray of lipid rafts as a form of tethered bilayer lipid membranes (tBLMs). Nanopatterns of UV-curable PVA were fabricated on gold substrates by ultraviolet nanoimprint lithography (UV-NIL). The surface of PVA hydrogel strongly restricted the adhesion of vesicles, resulting in selective arrays into the nanowells (NWs) of PVA hydrogel. A vesicle fusion and a mixed self-assembled monolayer (SAM) assisted vesicle fusion methods were used to confine the tBLM-formed lipid rafts into the nanopatterned PVA hydrogel. The tBLMs were not formed into the PVA hydrogel NWs by a simple vesicle fusion method. Nanoarray of tBLM-formed lipid rafts into PVA NWs has been accomplished by raft vesicle fusion onto a mixed SAM comprised of a mixture of 2-mercaptoethanol and a PEG lipopolymer. We expect that nanoarrayed lipid raft as a form of tBLMs provides an alternative platform for the analysis of transmembrane proteins, membrane-associated proteins, cell adhesion and a useful tool enabling the high-throughput applications such as nanodevices or nanochips.

Our group has identified twenty-first aminoacyl-tRNA synthetase-suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes (E. coli GlnRS/E. coli tRNAGln, or Human initiator tRNA derivates, Kowal et. al., 1998 and Drabkin et. al., 1996). My research goal is to identify GlnRS mutants that can utilize the keto-containing glutamine analogue 2-amino-5-ketohexanoic acid (wherein amide amine group of glutamine is replaced by methyl group). The ketone group can undergo a variety of reactions including addition, aldol, and transamination reactions. It reacts with hydrazides, alkoxyamines, and semicarbazides under aqueous, mild conditions to produce hydrazone, oxime, and semicarbazone linkages that are stable under physiological conditions (Wang and Schultz, 2005). This important functional group is absent from the side chains of the common amino acids. To genetically encode this functional group in yeast in the form of 2-amino-5-ketohexanoic acid, I am working on isolation of GlnRS mutants.

The L-valine producing strain of Escherichiacoli was constructed by rational metabolic engineering and stepwise improvement based on transcriptome analysis and insilico gene knock-out simulation. The engineered L-valin producing base strain harboring pKKilvBN produced 1.31 g/liter L-valine. Comparative transcriptome profiling combined with in silico geneknock-out simulation was used for the enhanced production of L-valine. The VAMF strain (Val ΔaceF Δmdh ΔpfkA) harboring pKBRilvBNCED and pTrc184ygaZHlrp was able to produce 7.55 g/liter L-valine from 20 g/liter glucose, resulting in a high yield of 0.378 g L-valine per g glucose. The approaches described here can be a good example of developing strains for the enhanced production of amino acids based on systems biotechnology. [This work was supported by the Korean Systems Biology Project of the Ministry of Science and Technology (M10309020000-03B5002-00000). Further supports by the LG Chem Chair Professorship and KOSEF through the CUPS are appreciated].

Room temperature ionic liquids (ILs) have recently been very popular as green solvents due to their unique physicochemical properties of negligible vapot pressure, non-flammability, excellent thermal stability and a strong ability to dissolve a wide range of organic and inorganic compounds1). They have great potential as reaction media or co-solvent for biocatalysis and biotransformation2). There are many examples where the use of ILs improves activity, selectivity, and stability of enzyme. After giving a short overview on the application of ILs in biotechnology, the following topics will be covered in the presentation: Biocatalysis in ILs, bioseparation in ILs, recovery of ILs and toxicity of ILs.

In modern science and technology, size matters, because materials exhibit new characteristics in nano scale, which do not exist in bulky state. Scientists have made great efforts to utilize the distinctive properties of nano sized materials especially for overcoming drawbacks of conventional sensors. Quantum dots are one of representing labeling nanoparticles which have photostability, size tunable color, broad exciting energy level and narrow emission spectrum. Other noticeable labeling agents are gold nanoparticles because of their high light absorbing efficiency. Therefore, I investigated the use of the strong optical signaling nanoparticles as labeling agents for novel nanoscale materials such as DNA, proteins and carbon nanotubes. In particular, the nanoparticles were applied to develop simple homogenous assay modes. For these purposes, FRET was implied to both quantum dots and gold nanoparticles. FRET is controllable by engineering the interaction between targets and probe molecules. Molecular beacon and isothermal cycling probe technology were applied to engineering FRET on the nanoparticles for target specific signaling. Accordingly, I will presents the target detecting functions of the labeling nanoparticles and discuss functional effecting factors.

The kinetic properties required for sensitive and rapid sandwich ELISAs were determined rationally based on the kinetic model. And the screening conditions for isolation of target antibodies from a phage display library was designed based on simulation. The solid phase antibody are required to possess a sufficiently low koff value to minimize dissociation of the triple complex of the solid phase antibody, the antigen and the enzyme-conjugated antibody during the reaction in the solution of the conjugated antibody. Both the solid phase and the conjugated antibodies should possess high kon values to ensure a rapid reaction during a limited reaction time. However, with a decrease in koff value and with an increase in kon value, the appearance frequency in a repertory of antibody decreases1). Thus, the screening conditions were designed so that the most desirable antibodies would be isolated from a given diversity. Under the designed conditions, antibodies with the required kinetic properties were isolated successfully. Furthermore, we showed that the best combination of both antibodies for sandwich ELISA can be determined based on the kon and koff values of the candidate antibodies. Our rational strategy would contribute to rapid development of sensitive ELISAs.

To display β-galactosidase whose bacterial surface display is not easy due to its tetramer formation and large size (116 kDa), Bacillus subtilis spore display system was developed. Among Bacillus subtilis spore coat proteins, eleven coat proteins were examined, and CotE and CotG were selected. SDS-PAGE analysis of proteins extracted from purified spore of Bacillus subtilis, FACS analysis of purified spore, protease accessibility test proved successful surface display of β-galactosidase on Bacillus subtilis spore. β-galactosidase displayed on Bacillus spores was used as a whole-cell immobilized biocatalyst for transgalactosylation in water-solvent biphasic reaction systems. Successful display of GFPUV and streptavidin using Bacillus subtilis spore can be used for various biotechnological application.

I will present our recent findings on the pathways and their regulation involved in central carbon metabolism in the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. The entire genome sequence of this archaeon has been determined (1) and gene disruption systems have been developed (2). A whole genome DNA microarray is also available for transcriptome studies. T. kodakaraensis can utilize a variety of organic compounds for growth, which include poly(oligo)saccharides, peptides, amino acids and pyruvate (3). This archaeon utilizes the modified Embden-Meyerhof pathway for glycolysis, and we have examined the enzymes involved in the conversion of glyceraldehyde 3-phosphate to 3-phosphoglycerate, and phosphoenolpyruvate to pyruvate (4). In terms of gluconeogenesis, we have performed a biochemical and genetic analysis on the key enzyme fructose-1,6-bisphosphatase (5). Enzymes involved in the production of glycogen have also been identified (6), as well as a novel degradation pathway for chitin (7). T. kodakaraensis does not harbor a complete set of genes corresponding to the pentose phosphate pathway, necessary for pentose and nucleotide synthesis in bacteria and eukaryotes. We instead found that a reverse flux of the ribulose monophosphate pathway is responsible for pentose synthesis in this organism (8). Through investigations on the physiological role of Type III Rubiscos (9), we have identified a novel pathway that may be involved in retrieving excess pentose carbon and redirecting it back to glycolysis (10). In terms of amino acid metabolism, a variety of aminotransferase and 2-oxo acid:ferredoxin oxidoreductase homologs are present on the T. kodakaraensis genome. In search of (ADP-forming) acyl-CoA synthetases that should function in further breakdown of amino acids, we found that the five acetyl-CoA synthetase paralogs present on the genome encode acyl-CoA synthetases with distinct substrate specificities (11). A fruitful collaboration has also led to the identification of the regulator that is responsible for controlling glycolytic and gluconeogenic modes of growth in T. kodakaraensis (12).

Diabetic nephropathy (DN) is a renal disease which develops as a consequence of diabetes mellitus (DM). Microalbuminuria is the earliest clinical sign of DN. There are no specific diagnostic biomarkers for DN other than microalbuminuria. However, microalbuminuria does not constitute a sole independent indicator for DN. Therefore, 2-DE was utilized to identify biomarkers for the more specific and accurate prediction of progressive nephropathy in type Ⅱ DM, via comparisons of the serum proteome in three experimental groups: type Ⅱ diabetes patients without microalbuminuria (DM, n = 30), with microalbuminuria (MA, n = 29) and with chronic renal failure (CRF, n = 31). Differentially expressed proteins in MA and CRF groups as compared to those in DM group were identified by ESI-Q-TOF MS/MS and confirmed by Western blotting. Notably, RBP4 and eGPx were measured by ELISA using DM and MA patient samples. The sensitivities and specificities (based on ROC curves) of RBP4 and eGPx were 84% and 83% respectively, and 88% and 73% respectively. Collectively, the results show that the three proteins identified in this study may constitute potential biomarkers for the diagnosis of type Ⅱ DN.

Trichloroethylene (TCE) is the most frequently detected groundwater contaminants. Recent researches have demonstrated that TCE elicits positive chemotaxis (attractive response) in certain bacterial strains, most of which are capable of degrading TCE in the presence of toluene. Chemotatic responses to TCE by these TCE-degrading bacteria are expected to overcome mass-transfer limitation that impedes bioremediation processes and increase speed of biodegradation processes. However, little is known about the mechanisms at the molecular level. Psedomonas aeruginosa PAO1 is strongly repelled by TCE. Genetic analysis of negative chemotaxis (repelled response) to TCE by P. aeruginosa PAO1 revealed that the methyl-accepting chemotaxis proteins (MCPs) PctA, PctB and PctC which were identified as MCPs for amino acid, serve as the major chemoreceptors for the negative chemotaxis to TCE. Furthermore, we found that the pctABC triple mutant of P. aeruginosa PAO1 was attracted by TCE. P. aeruginosa PAO1 possesses 26 potential mcp genes in its genome. Chemotaxis assays of a set of mutants containing deletions in 26 potential mcp genes revealed that mcpA (PA0180) is the chemorecepter for positive chemotaxis to TCE. McpA also detects tetrachloroethylene and dichloroethylene isomers as attractants. Moreover, Pseudomonas putida F1, which is capable of degrading TCE, grown in the absence of toluene fail to respond to TCE. We introduced mcpA into P. putida F1 and the strain was attracted by TCE even when grown in the absence of toluene. We also found the chemoreceptor for positive chemotaxis to TCE in P. putida F1.

Asymmetric synthesis using efficient chemical or biocatalyst is the most desirable approach to synthesize chiral compounds. Transaminase can be used for the purpose of the production of chiral amino acids and amines. In general, alpha-transaminase has very low equilibrium constant, so that asymmetric synthesis is relatively easy to be developed, whereas w-transaminase has rather high equilibrium constant, so that more rigorous limitations always exist for the similar asymmetric synthesis. To understand these limitations in depth, we have mechanistically studied the half reaction of the transamination reaction. To design better asymmetric synthesis process, variations of amine group donor substrates were attempted, and underlying criteria to select a proper partner(amine donor) were analyzed, and the results will be discussed. In addition, to exploit better and broad applications of transaminase for the synthesis of chiral amine compounds, few examples of newly screened w-transaminases for target products, especially the compounds with double chiral centers, will be discussed, and computational strategies for expanding the substrate spectra of such transaminases were further attempted using homology modeling and sequence analysis. Their evaluation results will be presented.

As the amount of genetic information exponentially increases due to recent advances in genome sequencing, the demand for a faster and more parallel expression system has become greater than ever. For this reason, cell-free protein synthesis has received renewed attention as an alternative method for high-throughput gene expression. In contrast to in vivo gene expression methods, where protein synthesis is carried out in the context of cell physiology and is surrounded by cell walls and membranes, cell-free protein synthesis provides a completely open system that allows direct access to the reaction conditions. At the same time, most of the cell functions other than protein synthesis need not be maintained in the cell-free system. Here we present our recent achievements in developing a highly efficient cell-free protein expression system. Through a series of systematic approaches to address the key issues affecting the efficiency of cell-free protein synthesis reactions, currently we can produce milligram quantities of proteins in a simple batch reaction. Based on this effective and robust platform for rapid production of recombinant proteins, we currently are seeking to combine the techniques of cell-free protein synthesis with numerous other disciplines.

Hemicellulose or cellulose-based biomass is the most abundant renewable resource in nature and composed of lignin and carbohydrate polymers containing glucose, xylose, arabinose, mannose and so on. As xylose is one of the major carbohydrates in hemicellulosic biomass, it becomes an attractive feed stock for the production of value-added compounds. Bioethanol is designated as ethanol produced from renewable biomass and can be used as an alternative fuel for automobiles. Microbial production of bioethanol from hemicellulose has been developed using bacteria and yeasts. S. cerevisiae , a traditional alcohol fermenting yeast, is unable to use xylose as a sole carbon source because of its deficiency in xylose metabolic enzymes including xylose specific transporter, xylose reductase and xylitol dehydrogenase. In this study, the xylose transport mechanism of recombinant S. cerevisiae was elucidated in kinetic and molecular levels. The zero trans -influx assays of glucose and xylose under high and low glucose conditions, and complementation experiments with sugar transporters were chosen for the investigation of xylose transport characteristics. In addition, the expression level of xylulokinase, the third enzyme in xylose metabolism, was modulated for the optimization of bioethanol production in recombinant S. cerevisiae .

Following the success of genome sequencing projects, attention has now turned to studies on the function and structures of proteins. Although cell-based expression systems have been widely used, they have certain limitations in view of the quality, quantity, or high-throughput production of proteins. Many of those limitations can be circumvented by the use of cell-free translation systems. In the past few years, advances in the use of cell-free system have been made. Among such systems, the wheat germ-based system is of special interest for its eukaryotic nature. Since the protocol bypasses most of the time-consuming steps, it may provide a powerful means of rapid and systematic screening in many applications, such as high-throughput enzymatic testing of a large number of gene products for functional annotation; construction of protein-protein interaction identification systems; high-throughput identification of the 3D structure through NMR or X-ray diffraction; and industrial-scale protein production systems.Here we will highlight a new high-throughput screening method for searching substrate proteins for modifying enzymes and for the antigen discovery adapted to modern analytical systems such as AlphascreenTM (ParkinElmer). The information accumulated on gene product function and structure should revolutionize understanding of biology and fundamentally alter the practice of medicine, possibly influencing other industries as well.

Optimal microbial strains for bioethanol fermentation must utilize various carbon sources and withstand stressful conditions such as high temperature, high osmotic pressure, high concentration of ethanol, and, in some cases, extreme pH. We have developed a method in microbial cells that uses combinatorial libraries of zinc finger–containing, artificial transcription factors to induce stress resistance and to identify genes involved in stress resistance. By linking multiple zinc finger domains together, we constructed more than 300,000 zinc finger proteins with varied DNA-binding specificities. These zinc finger proteins were fused to appropriate effector domains to produce libraries of transcription factors. Zinc finger transcriptional regulatory proteins, then, were expressed individually in yeast or in E. coli, and the transformed cells were screened for organic solvent tolerance and thermotolerance. Combining chromatin immunoprecipitation experiments and in silico prediction of target DNA binding sequences for the artificial transcription factors, we identified novel genes associated with the stress resistance in E. coli. Our results show that randomized libraries of artificial transcription factors are powerful tools for functional genomic studies and for microbial engineering.

P. stipitis, the best known xylose fermenting yeast, has been served as a gene source for metabolic engineering of xylose fermentation in S. cerevisiae. Specifically, three genes (XYL1, XYL2, and XYL3) coding for the xylose metabolic pathway enzymes (xylose reductase, xylitol dehydrogenase, and xyluokinase) have been introduced into S. cerevisiae. The resulting recombinant S. cerevisiae strains were able to assimilate xylose as a carbon source, but did not produce ethanol with high yields. Accumulated results from prior studies suggested that simultaneous perturbation of multiple genes might be required to facilitate high yield xylose fermentation in S. cerevisiae. In order to identify such a set of gene targets enhancing xylose fermentation, we investigated the levels of mRNA transcripts of P. stipitis grown under four different conditions through EST analysis. We discovered two notable features in the gene expression profiles. First, transcripts of glucose-6-phsphate dehydrogenase (GND1), transketolase (TKT1), and transaldolase (TAL1), involved in the pentose phosphate pathway, are strongly induced on xylose. Second, the expression of a NAD-specific glutamate dehydrogenase (GDH2) is elevated on xylose under oxygen limited conditions. These results suggest that higher capacity of the pentose pathway and a cofactor regeneration system are necessary for efficient maintenance of xylose metabolism.

Vibrio vulnificus, a human pathogen, exhibits a biofilm-forming activity and its ability requires global regulators, NtrC and RpoN. Both transcription factors regulate lipopolysaccharide (LPS) production and swimming motility, which are critically required for the initial stages of biofilm formation. In addition, exopolysaccharide (EPS) production is also influenced by these regulators. Thus, in this study, the relationships among the regulators, the genes encoding EPS biosyntheses, and their pathogenic roles were investigated to elucidate the complex but elaborate processes of biofilm formation by this pathogen. EPS production was highly induced under the nitrogen starvation conditions, and its induction required the presence of functional NtrC and RpoN. The gene clusters necessary for syntheses of EPS and capsular polysaccharide (CPS) were screened through in silico analysis of V. vulnificus genome sequences. Three clusters for EPS and a cluster for CPS were isolated, and their transcriptional characteristics were investigated. Only the EPS clusters were controlled by NtrC and RpoN, and their expression was significantly increased under the nitrogen starvation condition. The mutants deficient in one of the genes within each cluster showed decreased production of EPS and attenuated ability to form mature biofilm. Furthermore, these mutants showed significantly less adherence to the human epithelial cells than their isogenic wildtype, and one of the mutant exhibited decreased cytotoxicity to human cell lines. These results suggest that NtrC/RpoN-regulated EPS is the critical factors for biofilm formation and adherence of V. vulnificus

Enzymes are being widely used since it has many advantages, such as mild reaction conditions, substrate specificity. More than 3000 enzymes are known to work in cells and microorganisms, which means that enzymes can be used for 3000 different reactions in vitro. Especially, enzymes will play an important role in developing industrial biotechnology. Even though enzymes have enormous potentials for commercial applications in pharmaceuticals, food, bioelectronics, and chemicals industries, enzymes have also disadvantages to be solved; enzymes are in general expensive, the researches on enzyme applications have not yet been performed extensively, enzymes are not stable enough. Overproduction of enzymes using genetic engineering tools have been performed to reduce the enzyme cost. Screening of novel enzymes and improvement of enzymes using directed evolution and protein engineering have been performed to get better enzymes. Novel enzymatic reactions for bioenergy and chemicals production are currently very important issues nowadays. Immobilization of enzymes and novel enzyme reaction system are also regarded as important issues. In this presentation, the issues to be solved in enzyme engineering are presented and discussed along with recent efforts from academia and industries.

In the summer of 2006, a small group of researchers from three universities and a national energy laboratory with a common interest in biofuels met to discuss the formation of a collaborative research center. Driven by the high level of interest on the part of companies, government, and the public, rapid progress was made. The Colorado Center for Biorefining and Biofuels (C2B2) was announced at a press conference in March 2007 and began operations in July 2007. C2B2 is a partnership among Colorado State University, the University of Colorado in Boulder, the Colorado School of Mines, and the National Renewable Energy Laboratory. The combined research expertise within C2B2 involves more than 200 researchers with a wide range of specialties, including plant biotechnology; crop science chemical, biological, and thermal conversions; engines research; bioplastics; agricultural economics; greenhouse gas balances; environmental assessments; and sociological studies. Funding for the research and educational activities of C2B2 comes from more than 30 industrial sponsors as well as the State of Colorado and the participating universities. Research conducted through C2B2 may be "shared", with results and intellectual property available to all sponsors, or "sponsored", with results maintained confidential to a single sponsoring company. Through these projects, research opportunities are provided for undergraduate students, graduate students, and postdoctoral researchers. In this presentation, more information will be provided about the structure of C2B2 and opportunities provided by the center, and several of the ongoing research projects will be highlighted.

Bio-ethanol, which is one of the most studied renewable alternatives, can be obtained from the fermentation of sugar, starch, cellulose-based materials. Fermented beer from those raw materials contains 8～11 v/v % of ethanol and the beer is sent to distillation process so that 95～97 v/v % of hydrous ethanol can be produced. Although the ethanol from the distillation process is used for alcoholic beverage or industrial usage, its water content should be removed by an additional dehydration process such as azeotropic distillation or pressure swing adsorption process for using ethanol as blended fuel. In this study, an investigation was carried out on ethanol-water separation employing a PSA process with 3A zeolite as the adsorbent. The cycle of the PSA process was operated under the following variables: feed flow (80～100 L/h of 95 v/v% ethanol), adsorption temperature (110～140 ℃) and adsorption pressure (1.2 atm). PSA process including two molecular sieve dehydration columns filled with zeolites was installed at pilot plant scale for this study. The product purity through the dehydration columns reached minimum 99.5 v/v% and this PSA process could produce more than 1.0 kL of anhydrous ethanol on a daily basis.

Antifouling(AF) efficiency of a nontoxic aliphatic ester, ethyl heptanoate was evaluated against spores of a fouling alga, Ulva pertusa and a ship fouling diatom Amphora coffeaeformis. Based on the nontoxic AF activity exhibited in the laboratory bioassay, a hybrid Controlled Depletion Paint(CDP) formulation was prepared with ethyl heptanoate using acryl resin as a functional binder. To increase its performance, coatings were also prepared in combination with a booster biocide, zinc pyrithione(ZPT). Test panel studies were conducted with newly prepared hybrid CDP-AF coatings(ethyl heptanoate 10-40%) at Ayajin harbor, east coast of Korea from April 2005-May 2006. According to the fouling resistance recorded after 4, 8, and 13 months from the coatings, AF performance of the newly prepared hybrid CDP coatings are discussed.

The development of highly sensitive detection platforms is quite important to measure surface-generated signals due to the fact that biological signaling events as well as chemical adsorption behaviors generally occur on surfaces. Thus, a variety of surfaces fabrication methods have been applied to the sensitive and selective detection of biologically and environmentally relevant issues. Here, we present the effective monitoring methods via powerful surface analyzing techniques such as atomic force microscopy1-5) and surface plasmon resonance spectroscopy,6-8) which allows real-time observation of phenomena on surfaces. The effective detections of environmental pollutants were successively achieved by the fabrication of miniaturized electrode systems9-11) and self-assembled smart sensing layers.12-16) In addition, useful signals generated on surfaces were further amplified by some innovative methods, such as enlargement of surface area via mesoporous layer,17-18) multiplication of tiny signals via biomolecules19) and Au nanoparticles.20) The methods proposed here also provide the feasibility of intelligent monitoring for the environmentally and biologically meaningful issues.

Biofilm is a starting point of biofouling and/or biocorrosion in marine environment. However, only small information by multi-species is available. In the present study, properties of bacteria concerned in biofilm and interaction of selected bacteria during biofilm formation were investigated. Total 84 isolates from 3 days old biofilm could be assigned to 30 known genera which have been reported as widespread in marine environment. Approximately 31% of the isolates had been reported previously as uncultured or unidentified. Cell surface hydrophobicity and attachment ability were usually incompatible in a same strain and only 2 strains have both values high. Among the 39 strains produce Acyl-Homoserine Lactone (AHL), 21 strains could degrade AHL molecules. Strains belong to Actinobacteria displayed high cell surface hydrophobicity and Firmicutes displayed high attachment ability. In the continuous flow chamber system, growth-detachment- regrowth consist a cycle of biofilm process during 72 hours. It can be specified that, bacteria has high cell surface hydrophobicity plays major role at early stage and bacteria has high attachment attached ability plays after initial attachment.

The olfactory system plays an important role in recognizing environmental conditions. Since olfactory receptor genes were identified and cloned, various researches on olfactory systems have been carried out, and the interest in olfaction research has been increasing due to its potential industrial applications. In the smelling process, the binding of specific odorants to the olfactory receptor proteins is the initiation step in odor recognition and triggers signal transduction in a cell. Functional expression of the olfactory receptors on the surface of culturable cells would be very useful for application to an olfactory sensor. In this presentation I am going to discuss the method for an effective way to discriminate the odorants which bind to specific olfactory receptors. If the odorant molecules binding to each olfactory receptor are identified, this method can be applied to the olfactory biosensor. Several electronic noses have been developed; however, they have limitations in sensitivity and specificity. Array type devices coated with various cell lines expressing different olfactory receptors on each cell line would be a powerful tool for detecting and discriminating various odorants.

체내의 조직은 세포와 세포간, 세포와 세포간 물질의 상호작용으로 조성된 3차원적 환경을 이루고 있으며 이는 세포의 기능 유지에 매우 중요한 일익을 담당하고 있다. 따라서 손상된 조직이나 소실된 조직을 재생하고 복원하기 위하여서는 세포뿐 아니라 생체내의 환경을 모방하는 3차원적인 환경을 제공하는 것이 필요하다. ㈜리젠바이오텍에서는 천연고분자인 키토산과 합성고분자인 PLGA를 이용하여 3차원적인 지지체를 제조하고 다양한 인자들을 결합하여 생체내 혹은 생체외에서 조직을 복원하거나 재생하는 기술을 개발하고 있다. 또한 세포부착 단백질에대한 연구도 진행하여 조직재생 및 복원의 미세환경 복구 기술을 개발 중이다. PLGA 지지체는 현재 진피재생용으로 비뇨기과 제품으로 판매되고 있으며 연골재생용으로는 전임상 진행 중이다. ㈜리젠바이오텍에서는 개발된 PLGA 및 키토산 지지체를 이용한 다양한 조직재생에 대한 응용 기술을 연구 중이며 이와 같은 연구결과는 세포치료제 제품과 결합함으로써 치료제의 안정성 및 효능을 증진시킬 수 있으며 그 응용범위가 확대될 수 있을 것이다.

Although there are many methods known for detection of the protozoan parasite Cryptosporidium parvum in water, such as immunofluorescence or PCR techniques, they are labor-intensive and time-consuming sensor systems. To overcome these problems, label-free biosensors for this pathogen is highly desirable. Surface plasmon resonance (SPR) biosensor systems offer one alternative as label-free systems. A major advantage of SPR detection is that it does not require any labeling or staining of the analytes, and that the binding can be monitored in real-time.1,2) In this study, the SPR biosensor based on mixed self-assembled monolayers (SAMs) was developed for real-time detection of Cryptosporidium oocyst. To enhance the sensitivity of real-time detection, the modified flow type SPR biosensor was developed, which consisted of the immunoreaction step between the biotin-antibody and oocysts followed by the binding step of antibody bound oocysts onto the sensor surface. And also, a new real-time inhibition assay method was developed for detection of low concentration of C. parvum oocyst on the basis of flow type SPR biosensor.

당뇨병은 우리나라 전국민의 약 8%가 앓고 있고 최근 식생활의 서구화와 인구 노령화에 따라 그 환자 수가 크게 증가하고 있다. 말기 당뇨병환자의 15-25%가량이발이 썩어 들어가는 당뇨병성 족부궤양 (Diabetic Foot Ulcer) 환자이며, 이 병은 상처치유가 어려워 절단률이 20%가 넘는 당뇨병의 가장 심각한 합병증이다. 당뇨병성 족부궤양 세포치료제인 ‘하이알그라프트쓰리디 (Hyalograft 3D)’가 4년간 의 노력 끝에 식품의약품안정청으로부터 2007년 9월 14일 시판허가 되었다. 이 제품은 환자의 피부에서 채취한 진피조직으로부터 분리한 섬유아세포(Fibroblast)를 체외에서 3 주간 대량 증식 후 이를 다시 피부유사조직인 8 cm x 8 cm 크기의 히알루론산 유도체 Scaffold에 1 주일 정도 추가 배양하여 제조된 환자맞춤형 세포치료제이다. 이 세포치료제는 국내 최초의 세포복합형 조직공학 제품으로 다른 세포 치료제와는 달리 3차원 섬유상 Scaffold에 세포가 부착된 특징을 가지고 있어 두께가 있는 유사피부진피조직이라고 할 수 있다. 환자 자신의 세포를 이용하기 때문에 인체이식시 면역거부 반응이 전혀 없고, 살아있는 다량의 세포가 생체적합성이 뛰어난 히알루론산 Scaffold에 부착되어 인체에 이식되기 때문에 깊은 상처는 물론 상처부위를 흉터 없이 빠르게 재생시키고 재발율도 현저히 감소시킨다. 미국당뇨병학회지인 ‘Diabetes Care'에 게재된 논문에 따르면 이 치료제로 79명의 당뇨병성 족부궤양 환자를 치료한 결과 완치률이 65.3%에 이른다고 보고되는 등 이미 유럽에서는 피부궤양, 창상, 화상 환자들에게 널리 적용되어 그 안정성과 유효성이 입증됐으며 지금도 매년 2.000 여명 이상의 환자들이 치료받고 있다. 당뇨병성족부궤양 치료효과에 있어 단연 뛰어난 결과를 보여주는 ’하이알그라프트쓰리디‘는 손상된 피부조직을 송두리째 드러내야 하는 피부암이나 화상환자에게도 자신의 피부이식 없이 피부재생이 가능할 만큼 치료효과가 탁월하다. 이 세포치료제의 추가검증을 위한 시판 후 임상시험이 오는 10월부터 고려대 구로병원과 서울을지병원에서 실시될 예정이다.

Pseudomonas aeruginosa (P. aeruginosa) forms biofilm under the control of quorum sensing (QS). This study was designed to find out new QS inhibitors of P. aeruginosa, bearing N-3-oxododecanoyl-L- HSL (3OC12) in mind as one of the representative furanone based QS signals. Newly synthesized compounds as QS inhibitors which were a series of six new analogues of (5-oxo-2, 5-dihydrofuran-3-yl) methyl alkanoate were examined whether to disturb the autoinducer (AI, 3OC12)-LasR interaction of P. aeruginosa using E. coli DH5α co-transferred pJN105L and pSC11 plasmids. Each of the six synthesized compounds was confirmed using such techniques as NMR and mass spectroscopy. A bacterial test was performed to identify the activity of quorum-sensing inhibition and efficacy of biofilm control. Furthermore, the binding energy of these libraries in the receptor protein was estimated by FlexX program to elucidate the nature of the receptor protein-autoinducer (AI) interaction. All six synthesized compounds were shown to be QS inhibitors that interfere with AI-LasR docking of P. aeruginosa.

Recent advances in toxicity biosensing technologies have drawn heavy attentions from not only professionals but also public. One of the obstacles for this technique to be in full blossom is the relative weakness in securing biological contents having specific functions such as stress responses. In this presentation, as a type of biological contents, stress-specific bioluminescent bacteria are presented and discussed in terms of how they are developed and utilized as biological contents. The impacts of toxic chemicals on Escherichia coli could be analyzed using oligo DNA chips. The promoters of the specifically up-regulated genes from differential gene expression data of DNA microaray were inserted into a vector containing promoterless luxCDABE and transformed with E. coli strains, and numerous biosensing bacteria, therefore, have been finally constructed successfully. More than forty different recombinant bioluminescent bacteria having different promoters mentioned above were used for toxicity detection and classification through their specific bioluminescent response after fabricated in a form of cell array biochips (1, 2) or a multi-channel continuous mode. The fast toxicity detection and analysis is possible by using these biosensors/ biochips. These biosensors and biomonitoring systems could be implemented for high-throughput analysis of newly synthesized chemicals and drugs in addition to environmental toxicity analysis.

(주)엠씨티티는 2000년 원자력병원 손영숙박사에게서 이전 받은 ‘자가피부세포치료제’와 ‘키토산 인공진피’ 기술로 출발한 바이오벤쳐 기업이다. 엠씨티티는 무엇보다 skin wound repair 제품 개발에 주력하고 있으며, ‘자가피부세포치료제’로 케라힐 (Keraheal)을 제품화, 2005년 식약청으로부터 시판허가를 취득하였으며, 현재화상환자를 대상으로 임상 3상을 진행 중이다. 케라힐은 환자의 정상조직으로부터 채취한 작은 바이옵시(biopsy)로부터 피부세포를 분리하여 16일 동안 확장 배양후 바이알에 포장하여 환부에 뿌려주는 형태의 세포치료제이다. 기존의 자가피부세포치료제와 다른 점은 분화되지 않고 활발하게 증식 중인 피부각질세포로 이루어지고, 시트형이 아닌 현탁액의 성상을 가진다는 것이다. 케라힐 이외에도 현재 엠씨티티에서는 지방줄기세포를 이용한 주름개선치료제와인공 관절과 인공 각막에 관한 연구가 진행 중이며 배아줄기세포의 임상적용 가능한 세포주 수립 및 환경(niche) 재조합과 분화에 관한 연구가 진행 중이다. 엠씨티티는 성체 및 배아줄기세포에 관한 많은 연구 경험과 뛰어난 배양 기술을 바탕으로 향후 10년 내 세포치료제 분야에서 세계를 선도할 회사로 성장할 것이다.