Recombinant DNA technology and DNA sequencing

Abstract

DNA present in all our cells acts as a template by which cells are built. The human genome project, reading the code of the DNA within our cells, completed in 2003, is undoubtedly one of the great achievements of modern bioscience. Our ability to achieve this and to further understand and manipulate DNA has been tightly linked to our understanding of the bacterial and viral world. Outside of the science, the ability to understand and manipulate this code has far-reaching implications for society. In this article, we explore some of the basic techniques that enable us to read, copy and manipulate DNA sequences alongside a brief consideration of some of the implications for society.

Bioluminescence sensor: enzymes, reaction and utilization as an energy source

“…In its genome, this bacterium carries the genes encoding the luciferase enzyme called lux and the genes that enable the formation of the aldehyde substrate of this enzyme as an operon (lux operon, luxCDABE). In this operon, luxA and luxB genes encode subunits of the luciferase enzyme, while luxC, luxD and luxE genes encode elements of the multienzyme complex that provides the aldehyde substrate of lux luciferase from fatty acid [13,[15][16]. Today, skin, bone tissue, digestive tract, urinary tract, lung, tooth, middle ear infections are created experimentally on animals with many viruses, bacteria, parasites and yeast species modified with lux CDABE operon or other bioluminescence reporter genes.…”

Principles of synthetic biology

“…DNA lengths in the region of 3000–5000 bp are routinely made as standard, and some companies offer DNA synthesis up to 12 000 bp in length. In fact, the limiting factor for the length of DNA that can be synthesised is the length of DNA that can be accommodated by your chosen vector (see earlier essay in this series on recombinant DNA technology for more on DNA vectors [ 3 ]). M. genitalium JCVI-1.0 was assembled from synthetic fragments, 5000–7000 bp in length.…”

“…The cell components may be crude extracts, or they may be purified proteins, perhaps themselves made using bacteria or in a cell-free system. An example of a reaction performed in vitro with purified components is the polymerase chain reaction (PCR), used to amplify a DNA sample sufficiently to enable it to be studied (see earlier essay in this series [ 3 ]).…”

A Step‐by‐Step Guide for the Production of Recombinant Fluorescent TAT‐HA‐Tagged Proteins and their Transduction into Mammalian Cells

“…Thus, production of recombinant proteins represents a cornerstone for various applications in drug discovery (Andersen & Krummen, 2002; Overton, 2014), therapeutic proteins (Burnett & Burnett, 2020; Wurm, 2004) or vaccine production (Farzaneh et al., 2017; Joung et al., 2016). Recombinant DNA encoding proteins are typically generated through the use of recombinant DNA technology from historical methods (e.g., amplifications and use of restriction enzymes) to recently developed sophisticated technologies (e.g., Gibson assembly method for DNA cloning) (Bordat et al., 2015; Casini et al., 2015; Cohen, 2013; Ferro et al., 2019; Gibson et al., 2009; Jang & Magnuson, 2013; Kostylev et al., 2015; Li et al., 2019; Li et al., 2020; Li et al., 2018; Norris et al., 2015; Roberts, 2019; Rudenko & Barnes, 2018; Sands & Brent, 2016; Thomas et al., 2015; Wang et al., 2015). One of the most significant breakthroughs in recombinant protein technology has been the development of expression systems for the efficient production of complex proteins by selecting an appropriate method in microbial hosts (Brondyk, 2009), such as Escherichia coli (Baneyx, 1999; Baneyx & Mujacic, 2004; Chen, 2012; Gopal & Kumar, 2013; Rosano & Ceccarelli, 2014; Sørensen & Mortensen, 2005), yeast (Cregg et al., 2000; Mattanovich et al., 2012; Porro et al., 2005), Streptomyces spp .…”