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Microbial Lipase: Structure and Production
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
Yuji Shimada, Akio Sugihara, Yoshio Tominaga
The structural gene encoding P. fluorescens lipase was cloned into E. coli JM83 using pUC19 as a cloning vector [13]. This clone produced lipase, but the expression level was not very high. To overproduce the lipase in a recombinant E. coli, the lipase gene was inserted into an expression vector containing the tac promoter and rrnB transcription terminator. When E. coli harboring the recombinant plasmid was cultivated in the presence of isopropy 1-β-d-thiogalactoside (IPTG), it produced lipase as inclusion bodies. The amount of lipase reached more than 40% of the total cellular proteins. The inclusion bodies were solubilized by 8 M urea, and refolded into active lipase. This enzyme showed the same thermostability as the native lipase [60].
Basic Molecular Cloning of DNA and RNA
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
Competent cells are rather costly, and most labs prepare their own by setting aside one aliquot from a commercial batch and amplifying it. This is a recommended procedure but can be tricky. See Practical Tips 2.2 for recipes and tips for the preparation of chemically and electrically competent cells. Transformation efficiencies should be determined for each batch by using a standard plasmid, such as pUC19, at a known concentration. For routine cloning experiments, efficiencies of 106 transformants/μg are acceptable. However, for ligation reactions, mutagenesis, or other low-efficiency operations, values of 109–1011 are desirable. Some labs use homemade cells for routine operations and commercial cells for difficult cloning procedures.
The Use of Transient Electric Birefringence to Analyze Curvature in Naturally Occurring, Mixed‐Sequence DNA Molecules
Published in Stoyl P. Stoylov, Maria V. Stoimenova, Molecular and Colloidal Electro-Optics, 2016
Nancy C. Stellwagen, Yongjun Lu
Restriction fragments derived from the M13 origin and the VP1 gene in SV40 were obtained by digesting plasmid Litmus 28 (New England Biolabs) or the SV40 genome with suitable restriction enzymes. The desired restriction fragments were isolated by agarose gel electrophoresis, subcloned into the polylinker of plasmid pUC19, and amplified by standard methods [64]. After restriction enzyme digestion, the desired fragments were isolated by electrophoresis in 1% agarose gels, excised, and the agarose dissolved with a chaotropic salt (QIAquick gel extraction kit, Qiagen). The DNA fragments were then concentrated and desalted by adsorption on small DEAE columns, eluted into the desired buffer and stored at −20°C until needed. All fragments were sequenced to verify their identities.
ROS-mediated cell death induced by mixed ligand copper(II) complexes of l -proline and diimine: effect of co-ligand
Published in Journal of Coordination Chemistry, 2019
Sambantham Karpagam, Radhakrishnan Kartikeyan, Pappaiyan Paravai Nachiyar, Marappan Velusamy, Mani Kannan, Muthukalingan Krishnan, Upendra Chitgupi, Jonathan F. Lovell, Mohammad Abdulkader Akbarsha, Venugopal Rajendiran
The interaction of the complexes with SC pUC19 DNA was monitored using agarose gel electrophoresis. In reactions using SC pUC19 DNA, the plasmid DNA (SC form, 40 µM) in Tris-HCl buffer was treated with the copper complexes in the same buffer. In each experiment, the SC pUC19 DNA was treated with different concentrations of the complexes and also the cleavage of plasmid DNA in the absence and presence of the activating agent, ascorbic acid, was monitored using agarose gel electrophoresis. The samples were then incubated for 4 h in the absence of the reductant at 37 °C and analyzed for the cleavage products using gel electrophoresis as described below. Similarly, the samples were incubated for 0.5 h in the presence of a reductant. A loading buffer was added and electrophoresis performed at 50 V for 4 h in Tris-Acetate-EDTA (TAE) buffer (40 mM Tris base, 20 mM acetic acid, 1 mM EDTA) using 1% agarose gel containing 1.0 µg mL−1 EthBr. The gels were viewed in a gel documentation system and photographed using a CCD camera (Alpha InfoTech Corporation). The intensities of the SC DNA were corrected by a factor of 1.47 in view of the lower staining capacity of EthBr. The cleavage efficiency was measured by determining the ability of the complexes to convert the SC DNA (SC) to nicked circular (NC) form and linear form (LC).
Synthesis, characterization, theoretical simulation, and DNA-nuclease activity of a newly synthesized Mn–oximato complex
Published in Journal of Coordination Chemistry, 2018
Priyangana Deb, Madhulika Ghose, Nayim Sepay, Satyabrata Maiti, Kalyan K. Mukherjea
The double-stranded plasmid pUC19 DNA exists in a compact supercoiled (SC) form. Upon introduction of nuclease, the naturally occurring supercoiled (SC) form may give rise to nicked circular (NC) form. Relatively fast migration is observed for supercoiled (SC) form than the NC form when the plasmid DNA is subjected to electrophoresis. Hence, DNA-strand breaks were quantified by measuring the transformation of the supercoiled (SC) form into NC form [34]. The ability of the manganese complex to induce DNA-cleavage was studied by gel electrophoresis using supercoiled pUC19 DNA in Tris–HCl/NaCl buffer (pH 7.2) which was treated with increasing amounts of metal complex over a range of 18–36 µM along with H2O2 (16 µM) (Figure 8 and Table 4) [34, 35]. On addition of fixed concentration of H2O2 (16 µM), the percentage of NC form increases to 14% (lane 2), while on addition of the compound (18-36 µM), the percentage of NC form increases through 31% to 42% in lanes 3 and 5, respectively. Whereas, on addition of different concentrations of compound and H2O2 together (16 µM H2O2 + 18 µM complex) and (16 µM H2O2 + 36 µM complex) the percentage of NC form of DNA increases to 40% (lane 4) and 48% (lane 6), respectively. This clearly indicates that the compound alone shows moderate nuclease activity but exhibits higher nuclease activity when used in combination with H2O2 [22, 35].
A review on synthesis and applications of some selected Schiff bases with their transition metal complexes
Published in Journal of Coordination Chemistry, 2022
Attia Kanwal, Bushra Parveen, Rizwan Ashraf, Noman Haider, Kulsoom Ghulam Ali
Moubeen and coworkers studied mononuclear octahedral Ru(III) complexes with 2,2-((1E,1′E)-thiophene-2,5-diylbis(methaneylylidene))bis(azaneylylidene))diphenol Schiff bases. The anti-proliferative activities on human cervical cancer cells (HeLa) and breast cancer cells (MCF-7) were examined. All complexes had higher growth inhibition towards MCF-7 and HeLa cells; Ru(III) complex showed an IC50 against HeLa cells very close to that of cisplatin. Studies of the interaction with the CTDNA supported that these complexes bind DNA as demonstrated by gel electrophoresis studies. Ru(III) complexes had excellent efficiency to cleave the super coiled (SC) pUC19 plasmid DNA [47].