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Recombinant DNA technology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
A genomic library can be constructed by extracting the total genomic DNA of an organism. The DNA is broken into fragments of appropriate size, either by mechanical shearing, sonication, or by using a suitable restriction endonuclease for partial digestion of the DNA. Complete digestion is avoided, since it generates fragments that are too heterogeneous in size. For partial digestion, restriction enzymes having four-base (tetrameric) recognition sequences are employed in preference to those having six-base (hexameric) target sites. This is because a given four-base recognition site is expected to occur every 44 (=256) bp in a DNA molecule, while a six-base target site would occur only after every 46 (=4096) bp. (It is assumed here that the arrangement of the four bases in DNA molecules is random.) Therefore, the fragments produced in partial digests with enzymes having four-base recognition sites are more likely to be of appropriate size for cloning than those generated by enzymes having six-base recognition sites. Single or mixed digestions with the enzymes Alul, HaeIII, or Sau3A have been used for constructing genomic libraries. The use of restriction enzymes has the advantage that the same set of fragments is obtained from a DNA each time a specific enzyme is used, and many of the enzymes produce cohesive ends. The partial digests of genomic DNA are subjected to agarose gel electrophoresis or sucrose gradient centrifugation for separation from the mixture of fragments of appropriate size. These fragments are then inserted into a suitable vector for cloning. This constitutes the shotgun approach to gene cloning. In principle, any vector can be used, but A. vectors and cosmids have been the most commonly used, since DNA inserts of up to 23–25 kb can be cloned in these vectors. The vectors containing the inserts are cloned in a suitable bacterial host.
Development of tracking tool for p-nitrophenol monooxygenase genes from soil augmented with p-Nitrophenol degrading isolates: Bacillus, Pseudomonas and Arthrobacter
Published in Bioremediation Journal, 2020
Amol Nazirkar, Mayuresh Wagh, Asifa Qureshi, Ragini Bodade, Razia Kutty
However, very few studies have been carried out on tracking/monitoring the nitrophenol genes like PNP monooxygenases (PNP-MO) from environmental bacteria (Kutty, Purohit, and Khanna 2000; Kutty, Kapley, and Purohit 2001). Molecular biology techniques used in the field of bioremediation to monitor xenobiotic degrading bacteria are discussed in review published in 2002 (Widada, Nojiri, and Omori 2002). In 2010, there is a report on screening of genomic library for identification of PNP degrading gene cluster using a radio-labelled PCR product as DNA probe (Chauhan et al. 2010). Still, characterization of PNP monooxygenase encoding genes from these species of bacteria is lacking.
Cloning, overexpression, and structural characterization of a novel archaeal thermostable neopullulanase from Desulfurococcus mucosus DSM 2162
Published in Preparative Biochemistry & Biotechnology, 2022
Farzaneh Jafari, Farid Kiani-Ghaleh, Shahrzad Eftekhari, Mehdi Razzaghshoar Razlighi, Nazanin Nazari, Maryam Hajirajabi, Fatima Masoomi Sarvestani, Golnoosh Sharafieh
Desulfurococcus is a genus of thermophilic, anaerobic archaea with an optimal growth temperature of 85 °C and found in many hyperthermophilic environments. Desulfurococcus mucosus, the type species of the genus Desulfurococcus, was isolated from an acidic hot spring in Askja, Iceland by Zillig et al. with a validly published name in 1983.[21] Complete genome sequence of type strain O7/1 showed a total genome size of 1,314,639 bp length, containing 1,421 genes, 1371 protein-coding regions, and 50 RNA genes, with DNA G + C content of 53.1%.[22] In 2000, a gene, referred to as apuA, was isolated from D. mucosus genome and cloned in E. coli using a genomic library, subcloning, and expression experiments.[23] It was 1,974 bp in length and encoded a protein of 686 amino acids with an estimated molecular mass of 66 kDa introduced as a thermoactive pullulanase by Duffner et al.[23] The recombinant pullulanase (rapuDm) had an optimum activity at 85 °C and pH 5.0 and showed the following substrate’s preference: pullulan > amylose > starch > amylopectin > cyclodextrins, but not active on glycogen and dextran. In the present study, an annotated amylase gene (CP002363, Region: 748453.749880) from D. mucosus DSM 2162 genome, based on GenBank database, was amplified by PCR and cloned in E. coli to evaluate its possible expression and primary structural evaluation of the recombinant enzyme (DSMA). The purification of the recombinantly expressed enzyme and its biochemical characterization revealed that the DSMA is a new neopullulanase which is different from the previous pullulanase reported by Duffner et al.[23] Preliminary structural evaluations were performed using spectrofluorimeter, circular dichroism (CD) spectroscopy, and differential scanning calorimetry (DSC) to explain some structural features of the enzyme. Further, dynamic light scattering (DLS) analysis of the DSMA buffer solution at various concentrations of NaCl revealed its multimerization and/or aggregation behavior upon elevation of the ionic strength.