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Subsurface Processes
Published in Stephen M. Testa, Geological Aspects of Hazardous Waste Management, 2020
Any discussion of subsurface biological processes must include a newly recognized kind of genetic element called the plasmid. From the above discussion, it may seem as though the presence of enzymes to catalyze reactions with contaminants are random or fortuitous events based on normal cellular activity in the absence of the contaminant. However, the cell can play a more active role through the formation and use of plasmids. Plasmids are circular genetic elements that occur outside the chromosomes and reproduce independently of the chromosomes. Plasmids are not necessary for normal cell survival but have the ability to confer specific abilities to the cell, such as the biodegradation of specific compounds. This allows the cell to adapt to an environmental change, such as the presence of a new contaminant, without altering its intrinsic genetic code. Plasmids can be integrated into the chromosome to become permanent genetic material, but often disappear after the compound is no longer present. It is plasmids that have produced many of the organisms resistant to antibiotics, pesticides, etc. and it is obvious that plasmids will prove crucial to engineering subsurface microbes for biodegradation of many hazardous materials.
Cell Line Development
Published in Wei-Shou Hu, Cell Culture Bioprocess Engineering, 2020
Upon the introduction of the plasmid vector into the host cells, some cells will receive the vector. A portion of those vectors will enter the nucleus and express the encoded genes. Among the genes encoded in the plasmid is a selectable marker such as antibiotic resistance. The expression of the selectable marker allows the cell to survive in the presence of the selection reagent. Eventually, some plasmids that have entered the nucleus will be integrated into the genome and become a permanent part of the genome. Plasmids are not capable of self-replication, and free plasmids in the cell are gradually degraded. Only those cells that express the selectable marker will survive under selective conditions. Those that survive and grow under selection over time therefore have the vector, including the selectable marker gene, integrated into the genome. Many of the integrated vectors will also contain the GOI. The selectable marker gene and the selective condition, typically the presence or absence of the selective agent, are thus used in pairs. Some commonly used selectable markers and their mode of action in mammalian cells are listed in Table 6.1.
Advanced Methods of Degradation of Dyes in Textile Effluent
Published in Ram Lakhan Singh, Pradeep Kumar Singh, Rajat Pratap Singh, Recent Advances in Decolorization and Degradation of Dyes in Textile Effluent by Biological Approaches, 2019
Ram Lakhan Singh, Pradeep Kumar Singh, Rajat Pratap Singh
The first steps involved in GMM construction are the selection of dye degrading enzyme encoding genes, insertion into a vector and finally introduction into host cells. The vector is a genetic molecule used to transfer the new gene into other cells, where it replicates independently. Plasmids are most frequently used as cloning vectors in genetic engineering to multiply or express desired genes. A few genes encoding azoreductase were cloned from some bacteria, such as Bacillus stearothermophilus OY1-2 (Suzuki et al. 2001), Xenophilus azovorans KF46F (Blumel et al. 2002) and Escherichia coli (Nakanishi et al. 2001) to produce recombinant strains that would decolorize and degrade textile dyes.
Bioprocessing of recombinant proteins from Escherichia coli inclusion bodies: insights from structure-function relationship for novel applications
Published in Preparative Biochemistry & Biotechnology, 2023
Kajal Kachhawaha, Santanu Singh, Khyati Joshi, Priyanka Nain, Sumit K. Singh
Plasmids are the most common vectors utilized in recombinant DNA technology. Properties such as its size, copy number and promotor strength significantly contribute to the recombinant protein production in the host cell. The recombinant gene dosage is defined by the copy number of the plasmid. High gene dosage can either be favorable or unfavorable for the expression of the recombinant protein. Thus, a suitable plasmid should be selected, keeping it mind the characteristics it possesses.[56,57] To achieve a higher recombinant protein expression, the interest gene should be cloned immediately downstream to a strong promoter.[58] Strong transcriptional promoters control the foreign gene in various plasmids and bacteriophage vectors.[58] These promoters do not show constitutive expression and are governed by adding specific metabolites or changing culture conditions.[59] These are highly regulated in nature and also control the expression of the foreign gene in such a way that they do not interfere with the normal functioning of cellular genes and are not toxic to the host cell.[60] If these promoters are not regulated, they may result in loss of plasmid carrying the strong promoter or may be expressed constitutively, which shows detrimental effects on the cell.[59] The tae promotor is the most commonly used strong promotor.[58]
Production of bioactive recombinant monoclonal antibody fragment in periplasm of Escherichia coli expression system
Published in Preparative Biochemistry & Biotechnology, 2023
Preeti Saroha, Anurag S. Rathore
High copy number plasmids are related to increased protein production in bacteria.[3,21] However, these plasmids also overburden the cell and take a toll on cell metabolism and may even lead to cell death eventually, thereby resulting in low product yield.[9] Therefore, achieving appropriate cell growth and bioactive product yield while minimizing the stress on the cells would be desirable. In this paper, we report on soluble expression of antibody fragment against TNFα cultivating at low temperature using dual promotor vector without affinity tag and chaperones in E. coli (BL21 DE3). Combination of high copy plasmid number and low induction temperature yielded soluble TNFα inhibitor Fab up to 30 ± 3 mg/L in periplasmic region. We observed that soluble expression of recombinant TNFα inhibitor Fab was higher at low temperature (20 °C) when compared to 37 °C (Figure 5, Supplementary Figure S4, and Table 1). The mechanistic reason for this observation is that the level of ribosomes working on the peptide elongation of the proteins during translation is dependent on the growth rate which in turn is dependent on the cultivating temperature[22]. This may be plausibly attributed to decrease in the formation of in vivo aggregation due to slowing down of cellular synthetic machinery leading to reduced transcription and translation. Due to the reduced growth rate, nutrient uptake is reduced, thus curtailing the formation of toxic by-products.
A synthetic biology approach for the design of genetic algorithms with bacterial agents
Published in International Journal of Parallel, Emergent and Distributed Systems, 2021
A. Gargantilla Becerra, M. Gutiérrez, R. Lahoz-Beltra
Similar to genetic algorithms and other evolutionary algorithms, solutions are encoded in a string of characters. Since the agents simulate bacteria, the string represents a plasmid. Plasmids are circular strands of DNA that are different from chromosomal DNA. Present in bacteria, plasmids carry genes that give adaptive advantage to bacteria; when a bacterium reproduces by bacterial division each daughter bacterium receives a copy of the plasmid. Manipulation of plasmids allows designing customised gene circuits, being this one of the main goals of synthetic biology.