• Title/Summary/Keyword: Lambda Red recombination

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A Series of Vectors with Alternative Antibiotic Resistance Markers for Use in Lambda Red Recombination

  • Quick, Laura N.;Shah, Ashka;Wilson, James W.
    • Journal of Microbiology and Biotechnology
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    • v.20 no.4
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    • pp.666-669
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    • 2010
  • A target bacterial strain of interest for use in Red-based recombineering may already encode resistance to antibiotic markers used with current Red recombination tools, such that the resistance cannot be removed. Such cases include those where markers are needed to maintain an unstable genetic element co-resident in the strain or those where the genetic source of resistance is not known. We report the availability of PCR templates with FRT-flanked mutagenesis cassettes and plasmids encoding Red recombination functions that contain marker combinations not currently available on widely disseminated lambda Red molecular reagents. The functionality of these convenient alternative tools is demonstrated.

A Modified PCR-Directed Gene Replacements Method Using $lambda$-Red Recombination Functions in Escherichia coli

  • KIM SANG-YOON;CHO JAE-YONG
    • Journal of Microbiology and Biotechnology
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    • v.15 no.6
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    • pp.1346-1352
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    • 2005
  • We have developed a modified gene replacement method using PCR products containing short homologous sequences of 40- to 50-nt. The method required $\lambda$-Red recombination functions provided under the control of a temperature-sensitive CI857 repressor expressed from the $P_{lac}$ promoter in the presence of IPTG on an easily curable helper plasmid. The method promoted the targeted gene replacements in the Escherichia coli chromosome after shifting cultures of the recombinogenic host, which carries the helper plasmid, to $42^{\circ}C$ for 15 min. Since this method employs $\lambda$-Red recombination functions expressed from the easily curable helper plasmid, multiple rounds of gene replacements in the E. coli chromosome would be possible. The procedures described herein are expected to be widely used for metabolic engineering of E. coli and other bacteria.

Luminescence Characteristics of Mg2+·Ba2+ Co-Doped Sr2SiO4:Eu Yellow Phosphor for Light Emitting Diodes (LED용Mg2+·Ba2+Co-Doped Sr2SiO4:Eu 노란색 형광체의 발광특성)

  • Choi, Kyoung-Jae;Jee, Soon-Duk;Kim, Chang-Hae;Lee, Sang-Hyuk;Kim, Ho-Kun
    • Journal of the Korean Ceramic Society
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    • v.44 no.3 s.298
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    • pp.147-151
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    • 2007
  • An improvement for the efficiency of the $Sr_{2}SiO_{4}:Eu$ yellow phosphor under the $450{\sim}470\;nm$ excitation range have been achieved by adding the co-doping element ($Mg^{2+}\;and\;Ba^{2+}$) in the host. White LEDs were fabricated through an integration of an blue (InGaN) chip (${\lambda}_{cm}=450\;nm$) and a blend of two phosphors ($Mg^{2+},\;Ba^{2+}\;co-doped\;Sr_{2}SiO_{4}:Eu$ yellow phosphor+CaS:Eu red phosphor) in a single package. The InGaN-based two phosphor blends ($Mg^{2+},\;Ba^{2+}\;co-doped\;Sr_{2}SiO_{4}:Eu$ yellow phosphor+CaS:Eu red phosphor) LEDs showed three bands at 450 nm, 550 nm and 640 nm, respectively. The 450 nm emission band was due to a radiative recombination from an InGaN active layer. This 450 nm emission was used as an optical transition of the $Mg^{2+},\;Ba^{2+}\;co-doped\;Sr_{2}SiO_{4}:Eu$ yellow phosphor+CaS:Eu red phosphor. As a consequence of a preparation of white LEDs using the $Mg^{2+},\;Ba^{2+}\;co-doped\;Sr_{2}SiO_{4}:Eu$ yellow phosphor+CaS:Eu red phosphor yellow phosphor and CaS:Eu red phosphor, the highest luminescence efficiency was obtained at the 0.03 mol $Ba^{2+}$ concentration. At this time, the white LEDs showed the CCT (5300 K), CRI (89.9) and luminous efficacy (17.34 lm/W).

Biosynthesis of Polymyxins B, E, and P Using Genetically Engineered Polymyxin Synthetases in the Surrogate Host Bacillus subtilis

  • Kim, Se-Yu;Park, Soo-Young;Choi, Soo-Keun;Park, Seung-Hwan
    • Journal of Microbiology and Biotechnology
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    • v.25 no.7
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    • pp.1015-1025
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    • 2015
  • The development of diverse polymyxin derivatives is needed to solve the toxicity and resistance problems of polymyxins. However, no platform has generated polymyxin derivatives by genetically engineering a polymyxin synthetase, which is a nonribosomal peptide synthetase. In this study, we present a two-step approach for the construction of engineered polymyxin synthetases by substituting the adenylation (A) domains of polymyxin A synthetase, which is encoded by the pmxABCDE gene cluster of Paenibacillus polymyxa E681. First, the seventh L-threonine-specific A-domain region in pmxA was substituted with the L-leucine-specific A-domain region obtained from P. polymyxa ATCC21830 to make polymyxin E synthetase, and then the sixth D-leucine-specific A-domain region (A6-D-Leu-domain) was substituted with the D-phenylalanine-specific A-domain region (A6-D-Phe-domain) obtained from P. polymyxa F4 to make polymyxin B synthetase. This step was performed in Escherichia coli on a pmxA-containing fosmid, using the lambda Red recombination system and the sacB gene as a counter-selectable marker. Next, the modified pmxA gene was fused to pmxBCDE on the chromosome of Bacillus subtilis BSK4dA, and the resulting recombinant strains BSK4-PB and BSK4-PE were confirmed to produce polymyxins B and E, respectively. We also succeeded in constructing the B. subtilis BSK4-PP strain, which produces polymyxin P, by singly substituting the A6-D-Leu-domain with the A6-D-Phe-domain. This is the first report in which polymyxin derivatives were generated by genetically engineering polymyxin synthetases. The two recombinant B. subtilis strains will be useful for improving the commercial production of polymyxins B and E, and they will facilitate the generation of novel polymyxin derivatives.

Synthesis of Visible-working Pt-C-TiO2 Photocatalyst for the Degradation of Dye Wastewater (염료폐수 분해를 위한 가시광 감응형 Pt-C-TiO2 광촉매의 합성)

  • Hahn, Mi Sun;Yun, Chang Yeon;Yi, Jongheop
    • Clean Technology
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    • v.11 no.3
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    • pp.123-128
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    • 2005
  • Among various metal oxides semiconductors, $TiO_2$ is the most studied semiconductor for environmental clean-up applications due to its unique ability in photocatalyzing various organic contaminants, its chemical inertness, and nontoxicity. $TiO_2$, however, has a few drawbacks to be solved such as reactivity mainly working under ultraviolet irradiation (${\lambda}$ < 387 nm) and electron - hole recombination on $TiO_2$. In this study, to extend the absorption range of $TiO_2$ into the visible range and enhance electron - hole separation, we synthesized platinum (Pt) deposited $C-TiO_2$. The presence of Pt as an electron sink has been known to snhance the separation of photogenerated electron-hole pairs and induce the thermal decomposition. The characterization of as-synthesized $Pt-C-TiO_2$ was performed by Transmission Electron Microscopic (TEM), the Brunuer-Emmett-Teller (BET) method, X-ray Diffractometer (XRD), UV-vis spectrometer (UV-DRS), and X-ray Photoelectron Spectroscopy (XPS). In order to estimate the photocatalytic activity of the synthesized materials, the photoelectron Spectroscopy (XPS). In order to estimate the photocatalytic activity of the synthesized materials, the photodegradation experiment of an azo dye (Acid Red 44; $C_{10}H_7N=NC_{10}H_3(SO_3Na)_2OH$)was carried out by using an Xe arc lamp (300 W, Oriel). A 420 nm cut-off filter was used for visible light irradiation. From the results, Pt-deposited $C-TiO_2$ showed a far superior phothdegradation activity to Degussa P25, the commercial product under the irradiation of visible light and enhanced photocatalytic activity of visible-working $C-TiO_2$. This is a useful result into the application for the purification system of dye wastewater using visible energy of sun light.

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