• 제목/요약/키워드: kinetics of Cas9

검색결과 4건 처리시간 0.024초

A qPCR Method to Assay Endonuclease Activity of Cas9-sgRNA Ribonucleoprotein Complexes

  • Minh Tri Nguyen;Seul-Ah Kim;Ya-Yun Cheng;Sung Hoon Hong;Yong-Su Jin;Nam Soo Han
    • Journal of Microbiology and Biotechnology
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    • 제33권9호
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    • pp.1228-1237
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    • 2023
  • The CRISPR-Cas system has emerged as the most efficient genome editing technique for a wide range of cells. Delivery of the Cas9-sgRNA ribonucleoprotein complex (Cas9 RNP) has gained popularity. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR)-based assay to quantify the double-strand break reaction mediated by Cas9 RNP. To accomplish this, the dextransucrase gene (dsr) from Leuconostoc citreum was selected as the target DNA. The Cas9 protein was produced using recombinant Escherichia coli BL21, and two sgRNAs were synthesized through in vitro transcription to facilitate binding with the dsr gene. Under optimized in vitro conditions, the 2.6 kb dsr DNA was specifically cleaved into 1.1 and 1.5 kb fragments by both Cas9-sgRNA365 and Cas9-sgRNA433. By monitoring changes in dsr concentration using qPCR, the endonuclease activities of the two Cas9 RNPs were measured, and their efficiencies were compared. Specifically, the specific activities of dsr365RNP and dsr433RNP were 28.74 and 34.48 (unit/㎍ RNP), respectively. The versatility of this method was also verified using different target genes, uracil phosphoribosyl transferase (upp) gene, of Bifidobacterium bifidum and specific sgRNAs. The assay method was also utilized to determine the impact of high electrical field on Cas9 RNP activity during an efficient electroporation process. Overall, the results demonstrated that the qPCR-based method is an effective tool for measuring the endonuclease activity of Cas9 RNP.

Acid green-25 removal from wastewater by anion exchange membrane: Adsorption kinetic and thermodynamic studies

  • Khan, Muhammad Imran;Ansari, Tariq Mahmood;Zafar, Shagufta;Buzdar, Abdul Rehman;Khan, Muhammad Ali;Mumtaz, Fatima;Prapamonthon, Prasert;Akhtar, Mehwish
    • Membrane and Water Treatment
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    • 제9권2호
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    • pp.79-85
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    • 2018
  • In this work, batch adsorption of anionic dye acid green-25 (AG-25) from aqueous solution has been carried out at room temperature using anion exchange membrane (DF-120B) as a noval adsorbent. The effect of various experimental parameters such as contact time, membrane dosage, ionic strength and temperature on the adsorption of dye were investigated. Kinetic models namely pseudo-first-order, pseudo-second-order, Elovich, liquid film diffusion, Bangham and modified freundlich models were employed to evaluate the experimental data. Parameters like adsorption capacities, rate constant and related correlation coefficients for every model are calculated and discussed. It showed that adsorption of AG-25 onto DF-120B followed pseudo-first-order rate expression. Thermodynamic study indicates that adsorption of AG-25 onto DF-120B is an exothermic and spontaneous process.

$Co(OH)_2$로부터 수열법에 의한 코발트 분말제조 (Hydrothermal Reduction of $\Co(OH)_2$ to Cobalt Powder Preparation)

  • 김동진;정헌생
    • 한국재료학회지
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    • 제9권7호
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    • pp.675-679
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    • 1999
  • $PdCl_2$를 촉매제로 사용한 수소환원분위기에서 $Co(OH)_2$ 로부터 약 400nm크기인 구형의 코발트분말 제조에 관한 연구를 수행하였다. 본 실험에서 코발트의 환원반응속도는 표면반응 코어모델식에 잘 일치하였으며 이때 활성화 에너지는 $145~195^{\circ}C$에서 약 55.6KJ/mol 이었다. 또한 코발트의 환원속도는 초기 수소분압의 0.63승에 비례하는 가스화학흡착반응식으로 표시할 수 있다.

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STRAIN AND TEMPERATURE CHANGES DURING THE POLYMERIZATION OF AUTOPOLYMERIZING ACRYLIC RESINS

  • Ahn Hyung-Jun;Kim Chang-Whe;Kim Yung-Soo
    • 대한치과보철학회지
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    • 제39권6호
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    • pp.709-734
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    • 2001
  • The aims of this experiment were to investigate the strain and temperature changes simultaneously within autopolymerzing acrylic resin specimens. A computerized data acquisition system with an electrical resistance strain gauge and a thermocouple was used over time periods up to 180 minutes. The overall strain kinetics, the effects of stress relaxation and additional heat supply during the polymerization were evaluated. Stone mold replicas with an inner butt-joint rectangular cavity ($40.0{\times}25.0mm$, 5.0mm in depth) were duplicated from a brass master mold. A strain gauge (AE-11-S50N-120-EC, CAS Inc., Korea) and a thermocouple were installed within the cavity, which had been connected to a personal computer and a precision signal conditioning amplifier (DA1600 Dynamic Strain Amplifier, CAS Inc., Korea) so that real-time recordings of both polymerization-induced strain and temperature changes were performed. After each of fresh resin mixture was poured into the mold replica, data recording was done up to 180 minutes with three-second interval. Each of two poly(methyl methacrylate) products (Duralay, Vertex) and a vinyl ethyl methacrylate product (Snap) was examined repeatedly ten times. Additionally, removal procedures were done after 15, 30 and 60 minutes from the start of mixing to evaluate the effect of stress relaxation after deflasking. Six specimens for each of nine conditions were examined. After removal from the mold, the specimen continued bench-curing up to 180 minutes. Using a waterbath (Hanau Junior Curing Unit, Model No.76-0, Teledyne Hanau, New York, U.S.A.) with its temperature control maintained at $50^{\circ}C$, heat-soaking procedures with two different durations (15 and 45 minutes) were done to evaluate the effect of additional heat supply on the strain and temperature changes within the specimen during the polymerization. Five specimens for each of six conditions were examined. Within the parameters of this study the following results were drawn: 1. The mean shrinkage strains reached $-3095{\mu}{\epsilon},\;-1796{\mu}{\epsilon}$ and $-2959{\mu}{\epsilon}$ for Duralay, Snap and Vertex, respectively. The mean maximum temperature rise reached $56.7^{\circ}C,\;41.3^{\circ}C$ and $56.1^{\circ}C$ for Duralay, Snap, and Vertex, respectively. A vinyl ethyl methacrylate product (Snap) showed significantly less polymerization shrinkage strain (p<0.01) and significantly lower maximum temperature rise (p<0.01) than the other two poly(methyl methacrylate) products (Duralay, Vertex). 2. Mean maximum shrinkage rate for each resin was calculated to $-31.8{\mu}{\epsilon}/sec,\;-15.9{\mu}{\epsilon}/sec$ and $-31.8{\mu}{\epsilon}/sec$ for Duralay, Snap and Vertex, respectively. Snap showed significantly lower maximum shrinkage rate than Duralay and Vertex (p<0.01). 3. From the second experiment, some expansion was observed immediately after removal of specimen from the mold, and the amount of expansion increased as the removal time was delayed. For each removal time, Snap showed significantly less strain changes than the other two poly(methyl methacrylate) products (p<0.05). 4. During the external heat supply for the resins, higher maximum temperature rises were found. Meanwhile, the maximum shrinkage rates were not different from those of room temperature polymerizations. 5. From the third experiment, the external heat supply for the resins during polymerization could temporarily decrease or even reverse shrinkage strains of each material. But, shrinkage re-occurred in the linear nature after completion of heat supply. 6. Linear thermal expansion coefficients obtained from the end of heat supply continuing for an additional 5 minutes, showed that Snap exhibited significantly lower values than the other two poly(methyl methacrylate) products (p<0.01). Moreover, little difference was found between the mean linear thermal expansion coefficients obtained from two different heating durations (p>0.05).

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