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Medical biotechnology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
Molecular cloning refers to the process of making multiple copies of a defined DNA sequence. Cloning is frequently used to amplify DNA fragments containing whole genes, but it can also be used to amplify any DNA sequence, such as promoters, noncoding sequences, and randomly fragmented DNA. It is used in a wide array of biological experiments and practical applications, ranging from genetic fingerprinting to large-scale protein production. Occasionally, the term “cloning” is misleadingly used to refer to the identification of the chromosomal location of a gene associated with a particular phenotype of interest, such as in positional cloning. In practice, localization of the gene to a chromosome or genomic region does not necessarily enable one to isolate or amplify the relevant genomic sequence. In order to amplify any DNA sequence in a living organism, that sequence must be linked to an origin of replication, which is a sequence of DNA capable of directing the propagation of itself and any linked sequence. However, a number of other features are needed, and a variety of specialized cloning vectors exist, which allow protein expression, tagging, single-stranded RNA and DNA production, and a host of other manipulations. Cloning of any DNA fragment essentially involves four steps: (1) fragmentation, breaking apart a strand of DNA; (2) ligation, gluing together pieces of DNA in a desired sequence; (3) transfection, inserting the newly formed pieces of DNA into cells; and (4) screening/selection, selecting out the cells that were successfully transfected with the new DNA.
Trends in Biotechnology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2020
Molecular cloning by using recombinant DNA technology has been in use for making multiple copies of a single gene, and these multiple copies of genes are later used to make specific types of proteins. It has been observed that useful proteins such as human insulin can be produced by this technique. Presently, with the advent of synthetic biology, scientists can bioengineer microbes to perform complex multistep syntheses of natural products by assembling components of animal or plant genes that code for all of the enzymes in a synthetic pathway. Furthermore, synthetic biology techniques have been used to program yeast cells to manufacture the drug artemisinin, a natural product that is extremely effective in treating malaria. Currently, this compound can be extracted chemically from the sweet wormwood plant. The extraction of artemisinin is difficult and costly, which reduces its availability and affordability in developing countries of the world. Interestingly, researchers have found a method to reduce the cost of the drug by using a bioengineered metabolic pathway of yeast for the synthesis of precursor (artemisinic acid). Additionally, scientists have also assembled a group of several genes from sweet wormwood that code for the series of enzymes needed to make artemisinic acid, and inserted this cassette into the yeast Saccharomyces cerevisiae. Later on, the scientists analyzed the expression levels of each gene so that the entire multi-enzyme pathway can function competently. Once the engineered yeast cells have been coaxed into producing high yields of the artemisinin precursor, it becomes conceivable to manufacture this compound economically in large quantities by using fermentation technology. Moreover, a similar approach can be used to mass produce other drugs that are currently available in limited quantities from natural sources, such as anticancer drugs and the anti-HIV compounds.
Expression and characterization of cholesterol oxidase with high thermal and pH stability from Janthinobacterium agaricidamnosum
Published in Preparative Biochemistry & Biotechnology, 2023
Noriyuki Doukyu, Yuuki Ikehata, Taichi Sasaki
Janthinobacterium agaricidamnosum is a Gram-negative bacterium belonging to the phylum Proteobacteria.[22]J. agaricidamnosum is a mushroom pathogen that has been reported to cause soft rot disease in the cultivated mushroom Agaricus bisporus.[22] A COXase from pathogenic actinomycete Rhodococcus equi, which causes zoonotic infections in horses and foals, is thought to be a virulence factor due to its membrane-damaging property.[23] A gene sequence encoding a hypothetical COXase belonging to the VAO family has been deposited in the genome DNA sequence data of J. agaricidamnosum. In the present study, we report the molecular cloning of a COXase gene of J. agaricidamnosum, the expression and purification of the COXase, and the characteristics of the purified COXase.
Nanobodies targeting the interaction interface of programmed death receptor 1 (PD-1)/PD-1 ligand 1 (PD-1/PD-L1)
Published in Preparative Biochemistry & Biotechnology, 2020
Biyan Wen, Lin Zhao, Yuchu Wang, Chuangnan Qiu, Zhimin Xu, Kunling Huang, He Zhu, Zemin Li, Huangjin Li
The human domain antibody library (Source BioScience, Nottingham, UK) was used for phage screening. Escherichia coli DH5α (Merck, Darmstadt, Germany) was used for molecular cloning and E. coli BL21 (DE3) (Merck) was used as a host for human nanobody expression. The pET-21b vector (Merck) was used to clone the human nanobody gene. Ni-NTA, CM-Sepharose, Sephadex-G25, and Sephadex 75 columns were used for the purification of nanobodies (GE Healthcare, Buckinghamshire, UK). Human cervical carcinoma HeLa cells and human lung cancer A549 cells were purchased from the Chinese Academy Type Culture Collection. The human pancreas adenocarcinoma BxPC-3 cells and human mucoepidermoid pulmonary carcinoma NCI-H292 cells were purchased from the Chinese Jennio Biotech. DMEM, RPMI-1640, fetal bovine serum (FBS) and supplements were purchased from HyClone (Logan, UT). MPBS buffer is a PBS buffer supplemented with 5% marvel milk powder (w/v). PBST contained 0.1% Tween-20 in PBS buffer. 2 × YT medium was made by dissolving 16 g of bacto-tryptone, 10 g of yeast extract and 5 g of NaCl in 1 L of deionized water. Bacteria-tryptone (10 g), yeast extract (5 g), and NaCl (8 g) were dissolved in 800 mL of deionized water and mixed with 200 mL of 20% glucose solution (w/v). Ampicillin solution (1 mL) was then added to prepare TYE ampicillin glucose agar plates. For this, Ampicillin sodium salts were dissolved in deionized water to a concentration of 100 mg/mL and stored at −20 °C. PB consisted of NaH2PO4 and Na2HPO4 in deionized water.
Effects of microbial volatile organic compounds on Ganoderma lucidum growth and ganoderic acids production in Co-v-cultures (volatile co-cultures)
Published in Preparative Biochemistry and Biotechnology, 2019
Saeid Kalantari-Dehaghi, Ashrafalsadat Hatamian-Zarmi, Bahman Ebrahimi-Hosseinzadeh, Zahra-Beagom Mokhtari-Hosseini, Fahimeh Nojoki, Javad Hamedi, Saman Hosseinkhani
Culture composition is always among the first parameters for a research study examining the growth or metabolite production in any microorganism. And for G. lucidum, there are also many studies focusing on the effect of culture composition and different carbon sources.[5,7,59–61] On the other hand, several studies showed that mVOCs could serve as carbon and energy source, or even more specifically, supply precursors for biosynthesis pathways of the exposed microbe.[20,62–64] On the other hand, supplying precursor for GAs synthesis pathway must be considered as a potential mechanism for an increased amount of GAs by mVOCs in present co-v-cultures. As it has already been said, we believe that the wide range of influencing GAs production in co-v-culture could not come only from changes in media composition or oxygen concentration. Indeed, molecular studies and microarray-based analyses in several studies have shown that mVOCs profile from individual microbes can affect gene expression in exposed microbe with a different pattern.[9,43,51] Also, several studies have provided insight into GAs biosynthesis pathway through molecular cloning and quantifying gene transcription in G. lucidum