Earticle

We have selected aptamers binding to PAT protein from a DNA library by using SELEX (Systematic Evolution of Ligands of Exponential enrichment) method. At first, we carried out cloning of bar gene for expression of PAT protein in pGEX-4T-1 vector. Because of GST tagged protein, we used tag affinity chromatography method for obtaining of purified cloning PAT protein. For obtained herbicide-resistance PAT protein specific DNA aptamer, we performed 10 cycles of SELEX round and negative selection. We carefully selected GST protein for negative selection of herbicide- resistance PAT protein specific DNA aptamer. After that, we conducted 1 cycle of each round SELEX for selection of best round. We accomplished T-vector cloning to best round of SELEX for obtaining DNA aptamer candidates. Obtained DNA aptamer candidates were measured DNA concentration for selection of best DNA aptamer candidates by Nano-drop. Finally, we measured kd value between DNA aptamer to PAT protein by SPR (Surface Plasmon Resornance). A statistically oriented estimate of the significance of measured kd values showed tendency of PAT protein specific binding. A highly significant area for exploratory study of GMO detection system is very impressive area.

The autodisplay technology is one of the surface display methods for proteins or peptides at the E.coli outer membrane. In the autodisplay technology, proteins or peptides are expressed as recombinant proteins in a poly protein precursor for the autotransporter secretion pathway and automatically aligned on the outer membrane surface[1]. These aligned proteins or peptides can be used as a molecular recognition layer or a affinity layer for immunoassay[2]. For the preparation of outer membrane from autodiaplyed E.coli, lysozyme was treated to hydrolyze the peptidoglycan layer and triton X-100 was used for dissolving the cell membrane. After the outer membrane isolation, marker proteins from outer membrane, periplasm, inner membrane and cytoplasm were selected and analyezed by western blot and the enzymatic assay. OmpF, β-lactamase, secA and β-galactosidase were used for western blot and KDO, β-lactamase, NADH oxidase and β -galactosidase were analyzed by enzymatic assay. Isolated outer membrane were layered on the surface of 96 well microplate by using hydrophobic interaction[3] and layered outer membrane were analyezed by enzymatic assay. The purity of isolated outer membrane and its layer was calculated and the Z-domains, have a binding affinity to Fc region of IgG, autodisplaying outer membrane layer was used for the immunoassay to improve the sensitivity.

For a long time, enzyme-linked immunosorbent assay (ELISA) has been widely used for protein interaction asssay as well as various diagnostic systems. In conventional ELISA system, use of HRP conjugate antibody probes are required for detection signaling, however the low sensitivity of HRP reaction and use of high-cost antibody probes are considered as limitations. Recently various non-antibody scaffold proteins which have relatively small size (<100 aa) and rigid core structure have been developed as an alternative antibody. Compared with traditional antibody molecules, scaffold proteins have several beneficial points including i) simple production with high production yield by Escherichia coli host system, ii) easy engineering against various target materials with high affinity, and iii) high stability under the unfavorable environments which make scaffold proteins suitable for diagnostic system. Here, we developed a new fluorescence-linked immunosorbent assay (FLISA) system using GFP-fused scaffold protein allowing simple and sensitive detection of target molecules. In this system, GFP fused scaffold proteins could be produced in cytoplasm of E. coli with high productivity and solubility, and also successfully purified with high recovery yield. The purified protein maintained high affinity to the target molecules, and strong fluorescence intensity of GFP could be detected.

A human hybrid cell line named F2N78 was developed by somatic fusion of human embryonic kidney cells (HEK293) and Burkitt’s lymphoma cells (Namalwa). F2N78 cell line inherited advantageous phenotypes such as ease to suspension, high transfection efficacy, EBV genome insertion in chromosome, and human-specific glycosylation. For that reason, this cell line can be used for the production of human therapeutic antibody or vaccines. In order to be used in commercial processes, the formulation of the culture medium for F2N78 cell line has been investigated to optimize culture conditions. Glucose and glutamine are the most commonly employed carbon and nitrogen sources and directly affect cell growth. However, their rapid depletion was found to cause the accumulation of lactate and ammonia during F2N78 cell culture. To support cell growth with the generation of minimal quantities of lactate and ammonia, amino acids were chosen as the alternatives for glucose and glutamine. In previous studies, amino acids produced during cell culture were excluded from the culture medium. We calculated the percentage of amino acid consumption during cell culture and the results are as follows: Arg (50%), Asn (28%), Asp (100%), Cys (32%), His (29%), Ile (61%), Leu (70%), Lys (33%), Met (61%), Ser (89%), and Val (59%). These amino acids are selected for the design of MS004 medium by fortified amino acid study (FAS). In this study, effects of the addition of amino acids on cell growth of F2N78 cell line are going to be presented.

Actinomycetes are versatile producers of a variety of secondary metabolites, including more than two-thirds of all antibiotics employed in the fields of human medicine and agriculture. Among the known actinomycetes, only a few species are known to cause scab diseases in economically important root and tuber crops. Effective transformation procedure to facilitate molecular genetic studies of Streptomyces acidiscabies producing thaxtomin, which causes scab diseases in the economically important root and tuber crops was established via transconjugation from Escherichia coli ET12567 using an ØC31-derived integration vector, pSET152, harboring the oriT and attP fragments. Greatest number of exconjugants was achieved on MS medium containing 50 mM MgCl2 without heat treatment of spores and E. coli cells. Additionally, the integration site, attB, of the genome of S. acidiscabies exhibited the highest degree of homology with S. avermitilis and its chromosomal location was found to exist as a single attB site within an open reading frame coding for a pirin homolog similar to those identified in other actinomycetes. [This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology(2010-0008443)]

Transglutaminases (TGase) are a family of enzymes that catalyze an acyl transfer reaction between a γ-carboxyamide group of a glutamine residue in a peptide chin and a γ-aminogroup of a lysine residue, resulting in the formation of a ε-(γ-glutamyl) lysine cross-linkage. For applications of the food industry for the modification of proteins, the physiological properties of TGases derived from actinomycetes have been studied due to several advantages such as extracellular enzyme, calcium-independent, and general fermentation at low cost. For molecular genetics study of Streptomyces strains producing TGase, an effective transformation method was established based on conjugal transfer employing methylation-deficient E. coli ET12567(pUZ8002) as a DNA donor and using a phiC31-derived integration vector, pSET152, containing oriT and attP fragments. The optimal conditions for the conjugal transfer of Streptomyces strains producing TGase were searched. Additionally, the integration site, attB, in Streptomyces genomic DNA was cloned and characterized from several exconjugants. [This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (2010-0008443)]

Streptomyces natalensis produces natamycin, predominantly strong antifungal agent, inhibiting the growth of both yeasts and molds and preventing formation of aflatoxin in filamentous fungi. Due to the low toxicity of natamycin to mammalian cells compared to other antifungal compounds, it has found many applications in the treatment of various fungal diseases. Also, AfsR acts as a transcriptional factor in both the regulation of secondary metabolism and morphological differentiation in Streptomyces strains. In this study, PCR using the primers designed from the two highly conserved regions of the previously studied AfsR of Streptomyces strains gave 763- bp products. The sequence of this product had a high similarity to the expected region of an afsR gene. An intact 2,943 bp of afsR gene was cloned from S. natalensis by genomic Southern hybridization with PCR product as a probe, and then studied. To identify the function of the cloned afsR gene, it was inserted into pSET152ET to construct a high expression vector, and then was introduced into Streptomyces strains. The exconjugant including the high expression vector of afsR was constructed, and its phenotype was compared with the control strain. [This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (2010-0008443)]

Waste glycerol contains high amount fatty acids (~20%) as well as glycerol (~80%). Fatty acids are separated from waste glycerol and can be used for cosmetics or fine chemicals after treatment. The solution remaining is pretreated waste glycerol, which is fatty-acid free waste glycerol. This solution is toxic to microorganisms because it contains peroxide, methanol, and salts. This work explored the potential of using this pretreated waste glycerol to produce trehalose in E. coli by expressing trehalose biosynthetic genes (otsBA). Emerging areas of applications of trehalose are in pharmaceuticals as stabilizer of vaccines during storage, in cosmetics as a liposome stabilizer and in food industry as stable sweetener. In preliminary experiment, it was found that 37℃ was better than 27℃ for cell growth and trehalose production, and IPTG induction was essential. The Box-Behnken design (BBD) was applied for the optimizing concentrations of IPTG, validamycin A, and NaCl for trehalose production by engineered E. coli. Cells produced maximum trehalose (304 mg/L) at 0.1 mM IPTG, 10 μM validamycin A, and 298 mM NaCl. This result showed that pretreated waste glycerol can be used for the production of valuable products in engineered E. coli.

Cellulosic materials of plant origin are the most abundant utilizable biomass resource mainly made up of pentose and hexose sugars. In this study, an alcoholic fermentation from hemicellulosic substrate composed of pentose was carried out by recombinant Pichia pastoris expressing functional minicellulosome comprising scaffolding protein and hemicellulase. To increase hydrolysis efficiency, P. pastoris was engineered for the assembly of minicellulosome by heterologous expression of a recombinant scaffolding protein mCbpA and xylanase XynB from Clostridium cellulovorans. The resulting strain could degrade hemicellulosic substrate efficiently with synergic enzyme reaction. In the fermentation of hemicellulosic materials, approximately 3.87 g/L ethanol was produced and it was 1.3 fold higher than that of wild-type strain. This recombinant yeast strain was useful for direct ethanol production from hemicellulosic substrate with synergic effect of the hemicellulolytic cellulosome. Enhanced ethanol productivity was achieved at expressing both of scaffolding protein mCbpA and xylanase XynB. It was concluded that recombinant P. pastoris is very suitable for industrial ethanol production from hemicllulose.

Clostridium beijerinckii NCIMB 8052 as a butanol producer contains plenty of hydrolase and is able to utilize wide spectrum of carbohydrates. A 1,3-β-D-glucanase gene, which is annotated as Cbei_2828, was cloned from Clostridium beijerinckii NCIMB 8052 and expressed and purified in Escherichia coli BL21 (DE3). This glucanase gene has 1,362 base pairs and encodes a protein of 410 amino acids with a calculated molecular mass of 45 kDa. Signal peptideof this hydrolase was not cloned, so the total expressed protein has a molecular mass of 42 kDa. The encoded protein comprises a catalytic domain of glycosyl hydrolase family 16, fibronectin type 3 domain and Chitin/cellulose binding domain. The expressed enzyme was characterized. The activity of the expressed protein was tested by zymography and TLC by incubating the crude enzyme in Tris-Cl buffer containing 1% laminarin (w/v). The optimum working temperature and pH, substrate specificity and thermal stability of the purified enzyme will be tested. Kinetic parameters such as Km value and Vmaxwill also be evaluated.

In biopharmaceutical manufacturing, many products are produced by Chinese hamster ovary (CHO) cells. Validation of viral safety is essential in ensuring the safety of CHO cell culture-derived biopharmaceuticals, because CHO cells contain endogenous retrovirus like particles(RVLP). RVLP should be removed and/or inactivated during the manufacture of biopharmaceuticals, and must not be present in the final product. Therefore validated quantitative detection method is required for ensuring the safety of biopharmaceuticals against RVLP. In this study, a TaqManbased real-time quantitative PCR assay to detect RVLP was developed and validated. Specific primers for amplification of RVLP nucleic acid was selected, and RVLP RNA was quantified by use of TaqMan probe. The sensitivity of the assay was calculated to be 2.24×100 copies/μL. The TaqMan-based real-time PCR assay was proven to be reproducible and very specific to RVLP. The established real-time PCR assay was successfully applied to the detection of RVLP during the manufacture of a therapeutic antibody using CHO cells. Therefore, it was concluded that this fast, specific, sensitive, and robust assay is invaluable for detection of RVLP and virus clearance evaluation during the manufacture of CHO cell culture-derived biopharmaceuticals.

Large amount of glycerol production from biodiesel industries as byproduct has substantially decreased its cost. This attracted many fermentation industries to use glycerol as carbon source. The production of industrially important chemicals such as 1,3-propanediol (1,3-PDO), 3-hydroxypropionic acid (3-HP) etc. from glycerol will be an value added contribution to biodiesel industries. Citrobacter amalonaticus Y19, a chemoheterotrophic enterobacter well documented for its ability to convert carbon monoxide to biohydrogen was studied for its capability to assimilate glycerol as the sole carbon source [1]. C. amalonaticus Y19 when cultivated under controlled conditions in presence of glycerol produced significant amount of various industrially important metabolites such as 1,3-PDO, 3-hydroxypropanealdehyde, 3-HP, pyruvate and ethanol by consuming glycerol at high rate. The contig sequencing and genome annotation revealed the presence of glycerol assimilating genes similar to E. coli along with coenzyme B12 synthesizing and 1,2- propanediol degrading genes. Vitamin B12 synthesis and faster glycerol consumption rate made C. amalonaticus Y19 a more promising strain for 1,3-PDO production.

Industrialization has resulted in emission of large amount of carbon monoxide into the atmosphere, which is hazardous to living organisms. Therefore conversion of CO to non toxic materials is highly essential. Hydrogen production from CO will be a value added contribution in replacing fossil fuels, as hydrogen has enormous potential as future energy due to its abundance and non-polluting nature. Citrobacter amalonaticus Y19, a facultative anaerobic bacterium, isolated by Jung et al. (2002) [1], was characterized for hydrogen production from the toxic carbon monoxide gas. Contig genome analysis of this microorganism revealed that cooFSCTJhypB genes are involved in expressing functional carbon monoxide dehydrogenase (CODH). These genes were heterologously overexpressed in E.coli BL21 (DE3) by cloning in pACYCDuet vector. This resulted in an enzyme activity of 269 μmol CO min-1mg protein-1, which is comparatively higher than the CODH activity in wild type Y19 [2].

Lipase (EC 3.1.1.3) is a popular enzyme used as a biocatalyst for many biochemical reactions. Lipase is usually expressed in the Escherichia coli cytoplasm as an inactive inclusion body and at a low level. Cadida antarctica lipase B (CalB) was fused with various sizes of polyanionic amino acid tags and expressed in E. coli BL21 star (DE3) in order to improve the expression level of CalB and biological activity. The 5 aspartate tag (D5-CalB) attached at the N-terminal of the CalB gene resulted in the highest lipase activity of 3.3 U/ml in the medium, corresponding to a 8.2-fold enhancement compared with the authentic CalB. A fed-batch fermentation done with the recombinant E. coli grown in a pH-stat mode resulted in 2.6 g/L active CalB titer. Fusion of the 10 aspartate tag with the C-terminal of D5-CalB for purification (D5-CalB-D10) led to simple and effective purification of CalB with 89% purity and 72% purification yield.

Bacterial surface display finds its important biotechnological application in the fields of screening tools of evolved enzyme, bioremediation, whole cell bioconversion and tool for live vaccine production [1]. For the functional bacterial surface display of active enzyme of multimeric form, which is generally impossible due to molecular assembly of the monomer subunit subsequent to the secretion of displayed target protein outside the cell, a new surface display system based on Bacillus subtilis spore was developed. Among 11 spore coat proteins examined, cotE and cotG were selected. Using this motif, beta-galactosidase, which is active only in tetrameric form, was functionally displayed on the surface of Bacillus subtilis spore. The surface localization of beta-galactosidase was verified by enzymatic assay of purified spore, protease accessibility test and FACS analysis of spore expressing beta-galactosidase. While Bacillus subtilis spore wall integrity, examined by lysozyme and heat treatments, was slightly affected by the incorporation of CotE-LacZ fusion protein, it was not affected by the incorporation of CotG-lacZ fusion. Other enzymes such as streptavidin, lipase (lipB) of Bacillus subtilis and GFPUV were also successfully displayed using cotE and cotG motifs, and its possible application in various biotechnological fields will be discussed [2, 3]

Riboswitches are a powerful synthetic tool for gene regulation in molecular biology. They control gene expression by adopting alternative RNA structures in the presence or absence of small ligands. Most of the riboswitches are prokaryotic and here we describe a novel concept in gene regulation through eukaryotic intragenic riboswitch. In this investigation, we constructed a chimeric mRNA that contain theophylline aptamer after the start codon and studied the effect on translational gene expression in S.cerevisiae. It was observed on addition of theophylline, the strong secondary structure was formed at the mRNA 5’ proximal coding region caused by the theophylline aptamer, thereby disturbing the translation process. Consequently, it led to the decrease in the translational gene expression level. We also observed same effect using the tetracycline aptamer in this system. This new gene off-system can be used to simultaneously regulate the expression level of xylose reductase (XR) and xylitol dehydrogenase (XDH) from P.stipites, as well as reporter protein such as yellow fluorescent protein. The enzymes, XR and XDH are important in the bioethanol production by xylose fermentation and finding the optimal ratio of the enzymes in S.cerevisiae is critical for the success of the process. Our eukaryotic intragenic riboswitch can be used in addressing the bottle neck of xylose metabolic flux between xylose reductase and xylitol dehydrogenase in S.cerevisiae. This work was supported by the Advanced Biomass R&D Center (ABC-2010-0029737) through the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology.

Streptomyces sp. CSWu2 was recently isolated from Korean soil as a xylanase producer. An extracellular xylanase with 38 kDa molecular size was purified by a single step gel filtration using HQ-poros and various biochemical characteristics of the enzyme were determined. It showed high activity in neutral to alkaline condition and showed thermostabiity upto 60°C. It produced xylose and xylobiose as the major products from xylan. The activity was suppressed by most of the metal ions except potassium. The enzyme hydrolyzed corncob more efficiently than rice stem or rice husk. The thermostable and alkaline enzyme could be useful in various facets of biotechnology such as in xylooligosaccharides production, bioethanol production etc.

Streptomyces sp. CS802, recently isolated from Korean soil, produced xylanase in corncob medium. An extracellular xylanase (Xyn802) was purified by a single step gel filtration and biochemically characterized. It showed high activity in extremely alkaline condition with optimum pH at 12.0 and exhibited stability between pH 7.5 and 13.0. It produced xylobiose and xylotriose as the major products from xylan, suggesting its endoxylanase nature. N-terminal amino acid sequences of Xyn802 were ADRNANRD which are significantly different from the reported xylanase. The activity was enhanced by various detergents and a reducing agent and stable in range of organic solvents. Xyn802 produced by utilizing corncob, an agro-waste material, might be a novel xylanase based on its peculiar biochemical characteristics. Due to lack of cellulase activity and ability of producing xylooligosaccharides from xylan, Xyn802 can be a suitable candidate for pre-bleaching of paper pulps and generating xylooligosaccharides.

Industrial waste materials has become one of the global challenges and remained as a centre of attraction for the production of value added materials such as bioethanol and oligosaccharides. In an attempt to address the current situation, CS428, a new soil isolate capable of producing industrially applicable lignocellulose degrading enzyme, was isolated from Korean soil. An extracellular xylanase produced by the strain in culture medium containing 1.25% beech wood was purified and biochemically characterized. Enzyme production was highest at 2 to 3 days and the medium pH was found increasing with the increase in enzyme activity. The enzyme was thermostable and displayed broad range of pH stability (from pH 4.0 to pH 12.0). Further, the enzyme showed maximum activity at pH 12.0 and at 60°Cwith activity in broad temperature range (30 to 75°C). Because of high range of pH and thermostability, this enzyme can be a good candidate in the field of bioindustries.

In an attempt to isolate xylanase from microbial sources, Streptomyces sp. CSWu-1 was recently isolated from Korean soil and identified according to various characteristics including 16S rRNA sequencing. The strain produced high xylanase activity (XynWu-1) in xylan medium. Production profile showed that the highest activity was produced at 80 h when cultivated at 28⁰C and 120rpm. The enzyme was optimally active at 50°C and at pH 11.0, suggesting that the enzyme is alkaline and moderately thermostable. One peculiar characteristic of XynWu-1 is that it was highly dependent on buffer concentration and it maximum activity was found below 25mM.

Shigella genus and enteroinvasive Escherichia coli (EIEC) cause shigellosis whose symptoms range from just watery diarrhea to bloody diarrhea, fever and abdominal pain. Because as few as 10 of Shigella can cause infection, shigellosis is highly contagious. Also, S. sonnei is already predominant of developed countries and is becoming more prevalent in developing countries. Thus, early detection of S. sonnei is essential to prevent transmission of shigellosis. Up to now, various methods for the detection of S. sonnei have been reported based on polymerase chain reaction (PCR) and culture. However, these methods have limitation with time-consuming, laborious and potential of contamination. Therefore, development of rapid and accurate detection system for S. sonnei is required to prevent the spread of shigellosis. In this study, we carried out SELEX process to obtain IpaH specific DNA aptamer for early detection of S.sonnei. After 10 cycles of selection rounds, the binding affinity was confirmed by Nano-drop and SPR. The developments of IpaH aptamer can be applied to the early detection of S. sonnei.

The Growth rate for the biomass production of four microalgae, Chlorella sp. Dunaliella salina, Synechocystis w.t., Synechocystis Olive., were compared using the about 15L flat panel photobioreactors. The photobioreactor used in this study for decontamination and high concentration is composed of closed system. The photobioreactor is supplied with the constant rate of CO2 and air, Bubble-Airlift type and gas bubbling makes culture medium self circulation. Light source is generated by the white LED(light emitting diode) light guide plate which has broad band wavelength. Growh rate of culture species is measured by the optical density and the process of solid-liquid separation is used between culture medium and microalgae. The microalgaes are dried by freeze-dryer and dry weigth is measured. As a result of the experiment, Dunaliella salina's biomass productivities is 1.98g/L, the highest biomass productivities according to volume. Synechocystis w.t(1.5g/L), Chlorella sp.(1.35g/L)., Synechocystis olive(1.27g/L) are listed in order of low. Chlorella sp. is 0.1g/L/day, the highest growth rate for the biomass production considering culture period. Dunaliella salina(0.06g/L/day), Synechocystis olive (0.06g/L/day), Synechocystis w.t(0.04g/L/day) are listed in order of low.

Cellulose is a linear polymer of glucose units linked together by β -1,4-glycosidic bonds and is found as a major component of plant biomass that is naturally degraded by cellulolytic fungi and bacteria. Cellulose hydrolysis to glucose is performed via the synergic action of three cellulolytic enzymes: endo-β-D-glucanase (EC 3.2.1.4), exo-β- Dglucanase (EC 3.2.1.91) and β-glucosidase (EC 3.2.1.21). Production of glucose from agro industrial waste which can be utilized to produce value added products such as bioethanol is center of attraction of this era. Although there are several reports of potent cellulose producer from wide variety of microorganisms, it is rare from Streptomyces, which is known as the major producer of commercial antibiotics. In an attempt to screen Streptomyces strains capable of producing several cellulolytic enzymes from many Streptomyces strains preserved in our laboratory, caroboxymethyl cellulose (CMC)-agar plate method was used. Out of 62 strains, we found cellulolytic activity produced by 61 strains. Thus, although qualitative, we can conclude that almost all (~98%) Streptomyces strains are producer of cellulolytic enzymes in CMC-agar media, which can give a significant insight in the related field.

In the screening process of organic solvent tolerant bacteria showing good growth in media containing several kinds of refractory organic matter, one strain was isolated and identified as Bacillus subtilis. This study aimed to evaluate naphthalene degradation by Bacillus sp. BCNU 5006 isolated from a petrochemical sludge site. Naphthalene is a polycyclic aromatic hydrocarbon (PAH) that presents a high pollution potential and health risk and has been used as a model chemical for degradation studies on PAHs because of its relative toxicity. Optimal conditions for biodegradation of naphthalene were determined to be as follows: temperature 28°C, pH 7.0, inoculum size 0.2%. Under these conditions and initial naphthalene concentration of 100 ppm, more than 90% removal was obtained within 96 h. The strain was able to tolerate many organic solvents including benzene, toluene, xylene, octane, dodecane, butanol and ethylbenzene. Likewise, it could also utilize these solvents as the sole source of carbon with significant enzyme production. Consequently, above- mentioned findings suggest a potential bioremediation role of Bacillus sp. BCNU 5006 in the removal of naphthalene from wastewaters. (This work was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (Contract No 2010-0009141)).

Regulatory noncoding RNAs, such as small regulatory RNAs (sRNAs), are fast-acting and less-energy-consuming regulator widely used in many organisms to precisely control gene expression together with transcriptional control, and therefore they could be a vulnerable regulator for gene expression control. Here we present an approach for constructing synthetic small regulatory RNAs for controlling gene expression. We developed synthetic small regulatory RNAs repressing the translation of DsRed2 mRNA at various levels and also constructed three different sRNAs for the mRNAs of LuxR, AraC, and KanR without cross-reactivity. The results suggest that gene expression can be finetuned by designed artificial small RNAs. The possibility of designing small regulatory RNAs may facilitate the development of precisely regulated synthetic circuits. [This work was supported by the Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031963) funded by the Ministry of Education, Science and Technology, Korea and by the World Class University program (R32-2008-000-10142-0) of the MEST.]

Foot-and-mouth disease virus (FMDV) is the causative agent of an economically important disease that affects cattle and other clovenhoofed animals. VP1 is the most frequently studied protein owing to its significant roles in virus attachment, protective immunity, and serotype specificity. Here, we report high cell density cultivations of Escherichia coli for preparative scale production of FMDV VP1 in 5 L bioreactor. The epitope gene was expressed in cytoplasm of E. coli as GST fused format and its soluble expression (~ 90 %) was first confirmed by flask cultivation. After simple purification, antigenic activity of VP1 epitope was validated by western blot analysis and ELISA with commercially available monoclonal antibody. For large scale production pH-stat fedbatch cultivation was performed in 5 L jar bioreactor and various conditions were examined for higher productivities. Among examined conditions, when cells were induced at higher cell density (OD600=100) and complex feeding solutions were supplemented, significantly higher production yields could be achieved. The produced VP1 epitope can be used for development of protein therapeutics as well as diagnostic system.

Compaction of the bacterial nucleoid should not only allow for efficient replication and segregation during cell division, but it must also simultaneously participate in transcription and other processes involving the genome. The compact nucleoid body in a bacterial cell is extensively bound by several nucleoid- associated proteins (NAPs), which include HNS, HU, IHF, Fis, and Dps. A recent genome-wide analysis of NAPs association in E. coli genome identified several hundred binding regions but was limited in the ability to define binding motif. Here we improve on the resolution of chromatin immunoprecipitation coupled with massive sequencing (ChIP-seq) to determine the binding motif at single nucleotide resolution along with the distribution of the HNS-binding sites on a genome-scale. To this end, we optimized the ChIP-exo approach for bacterial applications, where an exonuclease trims ChIP DNA to a precise distance from the crosslinking site. Due to the ability to bridge adjacent tracts of DNA, HNS can locally induce some type of higher-order compaction, resulting in specific regulatory role in transcription. However, the molecular basis for the recognition of the local binding events has not been revealed. Our data enable the refinement of the HNS-binding motif in the DNA region bridged by HNS at single nucleotide resolution, providing a direct explanation for the mechanism underlying the transcription repression by HNS.

Synthetic biology aims to redesign biological systems with desirable properties to provide the foundation for the biosynthesis of metabolites and proteins of commercial interest. In this regard, Escherichia coli is one of the best industrial microbes. In an effort to supply only the necessary genes for full functionality, and therefore reduce the complexity of the metabolic and regulatory networks, large blocks of nonessential genes have been deleted1,2. However, removal of the nonessential genes resulted in reduction of the growth rate of the reduced-genome E. coli (MS56) on M9 minimal medium. To achieve a growth rate equivalent to that of wild-type E. coli K-12 MG1655, the reduced-genome MS56 strain was subjected to adaptive evolution on glucose M9 medium for 62 days. Along with achievement of the equivalent growth, the evolved E. coli strain (eJY62) exhibited unique physiological behaviors compared to the parental MS56 strain under the conditions, suggesting that the adaptive evolution resulted in significant metabolic and regulatory perturbations. To investigate the ability of the reduced-genome MS56 strain to overcome the reduction of growth rate, we monitored the acquisition and fixation of mutations that conveyed a selective growth advantage during adaptation to the M9 minimal medium by whole-genome resequencing. We identified 16 different mutations in the parental MS56 strain and evaluated the contributions of individual mutations to the reduced growth rate in M9 minimal medium. Several mutations identified from the eJY62 clones were responsible for the improved fitness of growth rates and the unique physiological properties. These results demonstrate that the E. coli can adapt to the genome reduction with several mutations, resulting in equivalent growth rate compared to the wild-type strain. This comprehensive genetic information on the reduction of microbial genome will provide the foundation for designing and rewriting an artificial genome.

PolyM-specific, polyG-specific and polyMG-specific alginate lyase were cloned by using PCR with homologous nucleotide sequences-based primers from various marine bacteria, previously. Overexpressed alginate lyase exhibited substrate specificity and showed low identity of protein sequence each other. The recombinant alginate lyases were homology modeled on the crystal structures as templates. Alginate oligomers produced from the recombinant alginate lyases with different substrate specificities were analyzed by FPLC and high-field 1H nuclear magnetic resonance (NMR) spectroscopy. Protein structures of substrate-specific alginate lyase were analyzed to have a b-jelly roll fold, ( α / α ) 6 barrel fold or parallel β-helix. Acknowledgement: This work was supported by New &Renewable Energy R&D program(20093020090020) and Korea Institute for Advancement in Technology (KIAT) through the Workforce Development Program in Strategic Technology under the Korea Ministry of Knowledge Economy(MKE).

A newly cloned alginate lyase from Sphingomonas sp. MJ-3 showed exo-type and edo-type alginate degrading activity. Homology-modelled structure of MJ-3 aginate lyase shared low relationship with reported crystal structures. When the multiple sequence and conserved domains were analyzed, interestingly, the cloned gene product was predicted to consist of AlgL (alginate lyase L)-like protein domain and heparinase-like protein domain. AlgL domain in N-terminal and heparinase domain in C-terminal were homology-modeled on 3AFL.pdb and 2FUQ.pdb as templates, respectively. The amino acid residues in proposed active site were mutated and characterized. Acknowledgement : This work was supported by New &Renewable Energy R&D program(20093020090020) and Korea Institute for Advancement in Technology (KIAT) through the Workforce Development Program in Strategic Technology under the Korea Ministry of Knowledge Economy(MKE).