China 
Africa 
Explore chapters and articles related to this topic
Enzymatic Synthesis and Modification of RNA Nanoparticles
Published in Peixuan Guo, Kirill A. Afonin, RNA Nanotechnology and Therapeutics, 2022
In order to complete enzymatic RNA synthesis, a DNA template is required for the polymerase enzymes to read and transcribe into RNA. Therefore, the desired sequence to be transcribed or produced must be designed in order to hybridize into the RNA nanoparticle upon completion of transcription. First and foremost, the DNA sequence must include the polymerase promoter sequence. Each polymerase has its own requirements to start transcription of a DNA strand; and when using the T7 RNA polymerase, a 17 nucleotide promoter sequence is needed to bind the polymerase to the DNA strand (Chamberlin & Ring, 1973a, Cheetham & Steitz, 1999). The end of the promoter sequence includes the “Tata box”, named due to its TATA sequence; immediately following the “Tata box”, the DNA sequence requires a GC or GG transcription start sequence in which the T7 polymerase will begin transcribing the rest of the DNA template sequence into RNA (Cheetham & Steitz, 1999). Therefore the enzymatic synthesis of the RNA is somewhat limited by sequence constraints as each strand must begin with either a GC or GG.
Enzymes Used for Recombinant DMA Technology Produced by Recombinant Microbes
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
They succeeded in overexpressing the T7 gene 5 using a T7 RNA polymerase-promoter system [40]. This system uses the unique specificity of T7 RNA polymerase for its own promoter to control the expression of other genes. The two compatible plasmids used for this system are shown in Figure 4. pGP5-3 contains the T7 gene 5 under the control of the T7 RNA polymerase promoter §10. pGPl-3 contains the gene for T7 RNA polymerase (T7 gene 1) under the control of the PL promoter, as well as the gene for the heat-labile repressor c/857. The two plasmids are maintained together in the E. coli strain by selection with ampicillin (pGP5-3) and tetracycline (pGPl-3). A shift of temperature to 42°C results in inactivation of the c7857 repressor, inducing the expression of T7 RNA polymerase. Transcription by T7 RNA polymerase initiating from the T7 RNA polymerase promoter on pGP5-3, in turn, results in the high expression of the T7 gene 5.
Cost-effective, high-yield production of Pyrobaculum calidifontis DNA polymerase for PCR application
Published in Preparative Biochemistry & Biotechnology, 2023
Kashif Maseh, Syed Farhat Ali, Shazeel Ahmad, Naeem Rashid
Heterologous gene expression in E. coli is a common method to produce recombinant proteins as it is a well-studied expression host with short doubling time and ability to express recombinant proteins at high rate.[19] In this study, we used E. coli BL21 CodonPlus (DE3) RIL strain for expression of Pca-pol gene. This strain contains rare-codon transfer RNAs. BL21 strain has also been reported to have better performance in high cell density cultures.[20]Pca-pol gene was cloned in plasmid pET28a. pET expression system is based on T7 promoter and requires T7 RNA polymerase from DE3 strain.[21] The resulting recombinant Pca-Pol contained N-terminal His-tag. Affinity tags such as His-tag facilitate the process of protein purification by affinity chromatography.[22] This can lead to greater recovery of the required recombinant protein, as in our case, IMAC purification recovered 92% of the total activity in the purified fraction.
Design of artificial cells: artificial biochemical systems, their thermodynamics and kinetics properties
Published in Egyptian Journal of Basic and Applied Sciences, 2022
Adamu Yunusa Ugya, Lin Pohan, Qifeng Wang, Kamel Meguellati
The top-down construction of ‘minimal cells’ is carried out by decreasing the genome of living cells. A primitive living organism does not require a high number of genes to be alive. Venter and colleagues discovered 517 genes in the parasitic bacterium Mycoplasma genitalium in 1995 [19]. An artificial infectious poliovirus was created by a top-bottom approach in 2002. With this method, the full-length poliovirus DNA (cDNA) is synthesized de novo and later transcribed into highly infectious viral RNA with the help of T7 RNA polymerase. Then, the transcription and replication of viral RNA took place in the cytoplasmic extract of uninfected cells, producing poliovirus with identical physiological and pathological properties compared to natural virus [20]. In 2004, minimal-gene sets were redefined for cell viability by Gil et al. [21]. Recently, a computer-based genome sequencing named Mycoplasma mycoides JCVI-syn1.0 was designed by Venter et al. on two strains of M. mycoides subspecies capri GM12 [22]. The expected phenotypic properties of M. mycoides, which can self-replicate, were seen in the resulting new cells. These types of cells are called ‘synthetic cells’ The developed synthetic DNA was integrated and accepted by the newly designed semi-synthetic artificial cell. Although the construction of large DNA sequences was enabled by synthetic biology, the creation of more complex artificial life still requires a long process to manipulate, modify, and develop them.
Metabolic engineered E. coli for the production of (R)-1,2-propanediol from biodiesel derived glycerol
Published in Biofuels, 2022
Wilson Sierra, Pilar Menéndez, Sonia Rodríguez Giordano
The E. coli FMJ39 strain, characterized as a ldhA, pflB1double mutant, unable to grow anaerobically on glucose was selected as the adequate genetic background for the present work. Supplying this strain with an alternative fermentation route will restore its ability to grow anaerobically on glucose or other carbon sources. The rationality behind the de novo design of an alternative metabolic pathway is depicted in Figure 1 and it involves the expression of two reductases able to sequentially reduce methylglyoxal (2) first to lactaldehyde (3) and then to (R)-1,2-propanediol (4a), yielding the desired product. However, E. coli FMJ39 strain was not suitable for heterologous protein expression from commercial T7 vectors, thus, inclusion of the T7pol-Gm-FRT cassette was necessary to afford the E.coli FMJ39T7pol-Gm-FRT strain. The strategy described by Kang and collaborators [50] was followed for construction of this strain by conjugating E. coli FMJ39 with the auxiliary strain E. coli (HPS1-mob-Δasd-pir116) (pBT20-Δbla-T7pol) that contains a plasmid carrying the sequence of the T7 RNA polymerase flanked with a transposon sequence. The new strain has the ability to express T7 RNA polymerase under the control of the lacUV5 promoter and provides a genomic background suitable for the abovementioned genetic manipulations.