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Metabolic Engineering of Nonmevalonate Pathway in Escherichia coli Enhances Lycopene Production

  • Kim, Seon-Won;J.D. Keasling
    • Proceedings of the Korean Society for Applied Microbiology Conference
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    • 2001.06a
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    • pp.141-145
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    • 2001
  • Isopentenyl diphosphate (IPP) is the common, five-carbon building block in the biosynthesis of all carotenoids. IPP in Escherichia coli is synthesized through the non-mevalonate pathway. The first reaction of IPP biosynthesis in E. coli is the formation of l-deoxy-D-xylulose-5-phosphate (DXP), catalyzed by DXP synthase and encoded by dxs. The second reaction in the pathway is the reduction of DXP to 2-C-methyl-D-erythritol-4-phosphate, catalyzed by DXP reductoisomerase and encoded by dxr. To determine if one or more of the reactions in the non-mevalonate pathway controlled flux to IPP, dxs and dxr were placed on several expression vectors under the control of three different promoters and transformed into three E. coli strains (DH5$\alpha$, XL1-Blue, and JMl0l) that had been engineered to produce lycopene. Lycopene production was improved significantly in strains transformed with the dxs expression vectors. When the dxs gene was expressed from the arabinose-inducible araBAD promoter ( $P_{BAD}$) on a medium-copy plasmid, lycopene production was 2-fold higher than when dxs was expressed from the IPTG-inducible trc and lac promoters ( $P_{trc}$ and $P_{lac}$, respectively) on medium-copy and high-copy plasmids. Given the low final densities of cells expressing dxs from IPTG-inducible promoters, the low lycopene production was probably due to the metabolic burden of plasmid maintenance and an excessive drain of central metabolic intermediates. At arabinose concentrations between 0 and 1.33 roM, cells expressing both dxs and dxr from $P_{BAD}$ on a medium-copy plasmid produced 1.4 - 2.0 times more lycopene than cells expressing dxs only. However, at higher arabinose concentrations lycopene . production in cells expressing both dxs and dxr was lower than in cells expressing dxs only. A comparison of the three E. coli strains transformed with the arabinose-inducible dxs on a medium-copy plasmid revealed that lycopene production was highest in XLI-Blue.LI-Blue.

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Empirical seismic vulnerability probability prediction model of RC structures considering historical field observation

  • Si-Qi Li;Hong-Bo Liu;Ke Du;Jia-Cheng Han;Yi-Ru Li;Li-Hui Yin
    • Structural Engineering and Mechanics
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    • v.86 no.4
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    • pp.547-571
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    • 2023
  • To deeply probe the actual earthquake level and fragility of typical reinforced concrete (RC) structures under multiple intensity grades, considering diachronic measurement building stock samples and actual observations of representative catastrophic earth shocks in China from 1990 to 2010, RC structures were divided into traditional RC structures (TRCs) and bottom reinforced concrete frame seismic wall masonry (BFM) structures, and the empirical damage characteristics and mechanisms were analysed. A great deal of statistics and induction were developed on the historical experience investigation data of 59 typical catastrophic earthquakes in 9 provinces of China. The database and fragility matrix prediction model were established with TRCs of 4,122.5284×104 m2 and 5,844 buildings and BFMs of 5,872 buildings as empirical seismic damage samples. By employing the methods of structural damage probability and statistics, nonlinear prediction of seismic vulnerability, and numerical and applied functional analysis, the comparison matrix of actual fragility probability prediction of TRC and BFM in multiple intensity regions under the latest version of China's macrointensity standard was established. A novel nonlinear regression prediction model of seismic vulnerability was proposed, and prediction models considering the seismic damage ratio and transcendental probability parameters were constructed. The time-varying vulnerability comparative model of the sample database was developed according to the different periods of multiple earthquakes. The new calculation method of the average fragility prediction index (AFPI) matrix parameter model has been proposed to predict the seismic fragility of an areal RC structure.

Case of construction method to the railway tunnel under the road (도로하부를 통과하는 철도터널의 특수공법 적용사례)

  • Kim, Sun-Hong;Jo, Yong-Seok;Kim, Dong-Ki;Park, Byung-Eun
    • Proceedings of the KSR Conference
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    • 2007.11a
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    • pp.337-344
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    • 2007
  • This paper is about the examination and selection of the special method applied to the railway tunnel which will lie under the road. Moreover, the road was constructed on the weak ground. Eventually, we select DSM, considering geological condition, stability during construction of the railway tunnel, and economic efficiency. The 3D-analysis, performed with PENTAGON-3D and MIDAS CIVIL-3D, shows that allowable standards are all satisfied. In the design of the railroad tunnel under the road at low depth, this paper will give useful data for the selection of the tunneling method.

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Mechanical features of cable-supported ribbed beam composite slab structure

  • Qiao, W.T.;Wang, D.;Zhao, M.S.
    • Steel and Composite Structures
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    • v.25 no.5
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    • pp.523-534
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    • 2017
  • Cable-supported ribbed beam composite slab structure (CBS) is proposed in this study. As a new cable-supported structure, it has many merits such as long span availability and cost-saving. Inspired by the previous research on cable-supported structures, the fabrication and construction process are developed. Pre-stress design method based on static equilibrium analysis is presented. In the algorithm, the iteration convergence can be accelerated and the calculation result can be kept in an acceptable precision by setting a rational threshold value. The accuracy of this method is also verified by experimental study on a 1:5 scaled model. Further, important parameters affecting the mechanical features of the CBS are discussed. The results indicate that the increases of sag-span ratio, depth of the ribbed beam and cable diameter can improve the mechanical behavior of the CBS by some extent, but the influence of strut sections on mechanical behavior of the CBS is negligible.

Flexural strength of roller compacted concrete pavements reinforced with glass-roved textiles

  • Madhkhan, Morteza;Nowroozi, Saeid;Torki, Mohammad E.
    • Structural Engineering and Mechanics
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    • v.55 no.1
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    • pp.137-160
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    • 2015
  • The one-way (two-way) flexural strength of RCC prisms (circular slabs) reinforced with glass fiber textiles is addressed. To this end, alkaline-resistant glass fiber textiles with three surface weights were used in the composite, the matrix concrete was designed with zero/nonzero slump, and the textiles were used with/without an intermediate layer provided by epoxy resin and sand mortar. Prisms were tested under a four-point loading apparatus and circular slabs were placed on simple supports under a central load. Effects of the amount and geometry of reinforcement, matrix workability, and the intermediate layer on the ultimate load and deflection were investigated. Results revealed that, with a specific reinforcement amount, there is an optimum textile tex for each case, depending on the matrix mix design and the presence of intermediate layer. Similar results were obtained in one-way and two-way bending tests.

Desulfurization of Dibenzothiophene and Diesel Oil by Metabolically Engineered Escherichia coli

  • Park, Si-Jae;Lee, In-Su;Chang, Yong-Keun;Lee, Sang-Yup
    • Journal of Microbiology and Biotechnology
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    • v.13 no.4
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    • pp.578-583
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    • 2003
  • The desulfurization genes (dszABC) were cloned from Gordonia nitida. Nucleotide sequences similarity between the dszABC genes of G. nitida and those of Rhodococcus rhodochrous IGTS8 was 89%. The similarities of deduced amino acids between the two were 86% for DszA, 86% for DszB, and 90% for DszC. The G. nitida dszABC genes were expressed in several different Escherichia coli strains under an inducible trc promoter. Cultivation of these metabolically engineered E. coli strains in the presence of 0.2 mM dibenzothiophene (DBT) allowed the conversion of DBT to 2-hydroxybiphenyl (2-HBP), which is the final metabolite of the sulfur-specific desulfurization pathway. The maximum conversion of DBT to 2-HBP was 16% in 60 h. Recombinant E. coli was applied for the deep desulfurization of diesel oil supplemented into the medium at 5% (v/v). Sulfur content in diesel oil was decreased from 250 mg sulfur/1 to 212.5 mg sulfur/1, resulting in the removal of 15% of sulfur in diesel oil in 60 h.

The Electrochemical Chlorination for Marine Plankton Community Disinfection (해양 플랑크톤 군집의 전기분해 염소소독 효과)

  • Kang, Jung-Hoon;Shin, Kyoung-Soon;Hyun, Bong-Gil;Jang, Min-Chul;Kim, Eun-Chan;Chang, Man
    • Journal of the Korean Society for Marine Environment & Energy
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    • v.10 no.3
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    • pp.127-137
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    • 2007
  • To confirm whether or not the Electrochemical Disinfection System (EDS) meet with the D-2 regulation established by IMO (International Maritime Organization), the biological treatment efficacy of the EDS was assessed using three groups of natural marine plankton (bacteria, $10-50\;{\mu}m$ and $>50\;{\mu}m$ sized organisms). Influent water was passed through the EDS under the flow velocity ($23.8\;m^3/hr$) and test design was consisted of control (no treatment) and experimental (10 ppm and 30 ppm) condition for total residual chlorine (TRC). And the biological condition of the influent water followed the standards established by the guidelines for the approval of ballast water management systems. The disinfection efficacy of the $10-50\;{\mu}m$ sized organisms (phytoplankton) was assessed by three kinds of measurements using photomicroscope, epifluorescence microscope and fluorometer (fumer Designs 10-AU). After being passed through the EDS, all motile phytoplankton lost their motility under photomicroscope, the colour of chlorophyll fluorescence fumed from red into green under epifluorescence, and the high chlorophyll fluorescence (Expt. 1: 6.95, Expt. 2: 7.11) detected by fluorometer decreased into value not detected. These results indicated phytoplankton community was totally killed after electrochemical disinfection treatment. Survivorship of the larger organisms than $50\;{\mu}m$ was determined based on the appendage's movement under a stereomicroscope. Natural assemblage collected from ambient seawater was killed shortly after being passed through the EDS, whereas some Artemia remained alive. However, no live Artemia was found after 24 hour further exposure to each TRC concentration (10 and 30 ppm) under darkness. After electrochemical treatment, the target bacteria such as aerobes, coliform and Escherichia coli were completely killed on the basis of CFU (colony forming unit) on Petrifilm plate ($3\;M^{TM}$) after 48 hr incubation. Moreover, no regrowth was found in the three groups of plankton during five days under additional exposure to the treated water. These results indicated that the disinfection efficiency of the EDS on the three groups of plankton satisfy D-2 regulation.

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Effects of Protein Kinase Inhibitors on Gene Activation of Early Embryos in Mouse (생쥐 초기배아의 유전자 활성에 미치는 Protein Kinase Inhibitors의 영향)

  • Lee, Jeong-Eun;Chai, Young-Gyu;Bae, In-Ha;Yoon, Young-Dal;Kim, Moon-Kyoo
    • Clinical and Experimental Reproductive Medicine
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    • v.22 no.2
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    • pp.191-201
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    • 1995
  • Transcriptional activation of the embryonic genome initiates at 2-cell stage in mouse embryo and is characterized by the synthesis of TRC which is restricted to 2-cell stage. To investigate the roles of various protein kinases on the embryonic gene activation, the effects of protein kinase inhibitors on in vitro development and protein synthetic profiles of the early mouse embryos were examinded. None of ${\alpna}-amanitin$ which is a mRNA synthetic inhibitor, H8 which is a PKA inhibitor, and H7 which is a PKC inhibitor, affected on first cleavage of mouse 1-cell embryos in vitro. However, all of these drugs inhibited the second cleavage. When the drugs were removed following treatment for 6 hours, H8 or H7 treatment showed little inhibition on subsequent development of 1-cell embryos to 2-cell stage or further. In contrast, ${\alpna}-amanitin$ irreversibly inhibited the development of 1-cell embryos to 2-cell stage following removal of the drug. Genistein, a TPK inhibitor, inhibited both the first cleavage of 1-cell embryos and the second cleavage of 2-cell embryos, suggesting that TPK activity may be important during the early cleavages. All of the above four drugs inhibited TRC synthesis as shown by the fluorographic analysis of $[^{35}S]-Met$ labeled protein profiles. When late 1-cell embryos were treated with H7 and analyzed synthetic patterns of $[^{35}S]-Met$ labeled protein, the quantitative differences of protein synthesis on SDS-PAGE appeared on 77 kD and 33 kD region at $32{\sim}38$ hours post hCG. From these studies, transcriptional activation of embryonic genome is not essenting to the mouse 1-cell embryos to develop to 2-cell stage. Hawever, TPK activity is reguisite for both the first cleavage and second cleavage. Similarly, both PKC and PKA activities are required for the second cleavage of mouse embryos, but not for the first cleavage.

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A Study on the Fabrication of the Composite Sabot for a Kinetic Energy Projectile (운동에너지탄용 복합재 이탈피의 제조에 관한 연구)

  • Choi, Jae-Ho
    • Journal of the Korea Institute of Military Science and Technology
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    • v.9 no.3
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    • pp.88-94
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    • 2006
  • In order to substitute current aluminum sabot and to increase the penetration performance of the kinetic energy projectiles, the research and development program for composites sabot has been conducted. For carbon/epoxy composites sabot, unidirectional carbon fiber reinforced epoxy prepreg was chosen and thick sectioned composites preforms with the different fiber angles along the circumferential direction of sabot were prepared by compression molding under the careful processing conditions at $150^{\circ}C$ for 1hour with $70kgf/cm^2$ curing pressure. The composites sabot demonstrated a weight reduction by approximately 30% than that of current aluminum sabot. The muzzle velocity of a kinetic energy projectile with composites sabot was measured to be about 63m/s higher than that with aluminum sabot. These results imply that the penetration performance is expected to be considerably increased when the composite sabot is applied to the kinetic energy projectiles.

A Molecular Biotechnology For Removal of Toxic Heavy Metals

  • Bang Sang-Weon;Clark Douglas S.;Keasling Jay D.
    • Proceedings of the Microbiological Society of Korea Conference
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    • 2000.10a
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    • pp.128-135
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    • 2000
  • The thiosulfate reductase gene (PhsABC) from Salmonella typhimurium was expressed in Escherichia coli in order to produce sulfide from inorganic thiosulfate and precipitate metals as metal sulfide complexes. A 5.1-kb DNA fragment containing the native phsABC and a 3.7-kb DNA fragment, excluding putative promoter and regulatory regions were inserted into expression vectors pTrc99A and pJB866, respectively. Upon expression of phsABC, E. coli DH5$\alpha$ harboring the phsABC constructs showed higher thiosulfate reductase activity and produced significantly more sulfide than the control strain (E. coli DH5$\alpha$) under both aerobic and anaerobic conditions. Among the four constructs, E. coli DH5$\alpha$ harboring pSB74 produced the highest level of thiosulfate reductase and removed most of heavy metals from solution under anaerobic conditions. In a mixture of 100 $\mu$M each of cadmium, lead, and zinc, the strain could remove $99\%$ of the total metals from solution within 10 hours. Cadmium was removed first, lead second, and zinc last. In contrast, a negative control did not produce any measurable sulfide and removed very little metals from solution. These results have important implications for removal of metals from wastewater contaminated with several metals.

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