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The Natural Versatility of RNA
Published in Peixuan Guo, Kirill A. Afonin, RNA Nanotechnology and Therapeutics, 2022
Lewis A. Rolband, Oleg A. Shevchenko, Caroline M. West, Ciara E. Conway, Caryn D. Striplin, Kirill A. Afonin
Riboswitches were discovered as a novel type of functional RNAs, having been first reported in 2002.65,66 They are untranslated segments of mRNA that induce changes to RNA structure to modulate gene expression. Riboswitches consist of two parts: a ligand-binding region and an expression platform. The ligand can range greatly in size and chemical makeup from a single ion to larger cofactors or peptides with high affinity. The ligand-binding region determines which class the riboswitch is categorized into, and there are currently around 40 known classes of riboswitches.65–68 The expression platform undergoes a structural change upon aptamer-target binding. This conformational shift in the expression platform causes alteration of gene expression by modulating transcription or translation.66–69 This modulation can be accomplished through several different mechanisms. Self-cleavage of the mRNA containing an internal riboswitch-ribozyme is an effective means of preventing a functional peptide from being translated.66–68 In some cases, a conformational shift will lead to the release or sequestration of the start codon or other ribosome binding sites, subsequently allowing or disrupting translation, respectively.68,70,71
Synthetic Biology: From Gene Circuits to Novel Biological Tools
Published in Tuan Vo-Dinh, Nanotechnology in Biology and Medicine, 2017
Nina G. Argibay, Eric M. Vazquez, Cortney E. Wilson, Travis J.A. Craddock, Robert P. Smith
Directly downstream of the promoter is the ribosomal binding site (RBS). The RBS is the region where the ribosome binds to the mRNA to initiate protein translation. By varying the DNA sequence of the RBS, one can control the translation initiation rate and thus the level of protein production (Salis, Mirsky, and Voigt 2009). Following the RBS lies the protein coding sequence, which contains the information encoding the protein to be produced. This area is often the most variable and most critical part of the gene circuit as it often directly participates in the desired behavior. Of particular note, some gene circuits will have multiple protein coding sequences downstream of a promoter. This allows parallel production of two or more proteins. In some instances, the first protein will be directly involved in producing the desired behavior. The second protein, often a fluorescent protein, will serve as an output of the promoter, thus verifying the activation of the promoter.
Thermostable Enzymes Produced by Recombinant Mesophilic and Thermophilic Bacteria
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
The nucleotide sequence of aldhT gene and the flanking regions were determined. A large open-reading frame (ORF) was found. It was composed of 1464 bp, corresponding to 488-amino acid residues. The Mr was estimated to be 52,912. The Shine-Dalgarno (SD) sequence, a probable ribosome-binding site, was located 9-bp upstream from the initiation codon (ATG). The typical prokaryotic promoter sequence [18,22] was located 33-bp upstream from the initiation codon. The nucleotide sequence resembling typical prokaryotic terminators was found downstream from the ORF. Homology of the primary structure was analyzed. The deduced amino acid sequence of the protein coded in this ORF showed significant homology (45%) to the human cytoplasmic aldehyde dehydrogenase.
Engineering Clostridium acetobutylicum to utilize cellulose by heterologous expression of a family 5 cellulase
Published in Biofuels, 2022
Mary Sanitha, Anwar Aliya Fathima, Andrew C. Tolonen, Mohandass Ramya
The cphy2058 (Cel5C) gene, including the ribosome binding site and secretion signal, were PCR amplified from C. phytofermentans genomic DNA using the forward and reverse primers 5 C Forward/Reverse (Table S2). The p5C plasmid was constructed by replacing the adc operon from C. acetobutylicum, which was initially present in the E. coli-Clostridium shuttle vector pSOS952 [9] between BamHI and NarI, with cphy2058. Expression of cphy2058 in p5C is driven by the C. acetobutylicum thiolase promoter flanked by lac operator sequences, and the adc terminator is downstream of cphy2058. The cphy2058 sequence in p5C was confirmed by sequencing. The resulting p5C plasmid was transformed into E. coli BL21(DE3) grown in Luria–Bertani medium supplemented with 1% glucose and transformant colonies were selected on ampicillin 100 µg mL−1. The cphy2058 gene in p5C was subsequently sequenced to confirm the gene and promoter sequences. Prior to electrotransformation into C. acetobutylicum, p5C was methylated in vivo using the pAN1 methylation plasmid [14]. C. acetobutylicum transformants with p5C, hereafter called DSM 792 (p5C), were selected with 40 µg mL−1 erythromycin and were maintained in 2X RCGM medium.
Protocatechuic acid production from lignin-associated phenolics
Published in Preparative Biochemistry & Biotechnology, 2021
Homologous gene vanAB encoding vanillate-O-demethylase from Pseudomonas putida KT2440 and Pseudomonas putida S12 (ATCC 700801) was PCR amplified and cloned along with the ribosome binding site in pSEVA 234 vector between the restriction site EcoR1 and BamHI. Similarly, synthetic heterologous gene vanAB encoding vanillate-O-demethylase from Acinetobacter sp. ADP1 was codon optimized for Pseudomonas putida and procured from GenScript. This gene along with the ribosome binding sequence (RBS) was cloned in between the restriction site EcoR1 and BamHI in pSEVA 234. Each of this plasmid construct was electroporated in the mutant strain of ΔpcaHG Pseudomonas putida KT2440 to evaluate the effect of gene expression on conversion of vanillic acid. The clones were selected on the cetrimide agar plate containing kanamycin (50 μg/mL).
Scale-up challenges and requirement of technology-transfer for cyanobacterial poly (3-hydroxybutyrate) production in industrial scale
Published in International Journal of Biobased Plastics, 2019
Donya Kamravamanesh, Daniel Kiesenhofer, Silvia Fluch, Maximilian Lackner, Christoph Herwig
PHB biosynthesis in cyanobacteria has been enhanced using optimization of cultivation conditions and multi-stage cultivation process which involves nitrogen or phosphorus limitation and the addition of sugars or organic acids, approaches which did not exploit the photosynthetic potential of cyanobacteria [4]. As an alternative way to increase productivity, cyanobacteria have been engineered, however, these attempts have shown little success. Recently, the optimization of the acetoacetyl-CoA reductase ribosome binding site in Synechocystis led to increase in (R)-3-hydroxybutyrate production of up to 1.84 g L−1 in 10 days from CO2 and the highest productivity of 263 mg L−1 d−1 was obtained [5]. As a substitute approach, random mutagenesis has also been used to obtain superior cyanobacterial strains in terms of growth and productivities. The authors previously showed the cyanobacterial strain MT_a24, a UV-mutated strain of Synechocystis sp. PCC 6714 produces PHB of up to 37 ± 4 % dry cell weight (DCW) under nitrogen and phosphorus limitation showing the highest productivity of 134 mg L−1 d−1 [6]. Under lab conditions, it was also shown that media optimization can be used to increase PHB content in MT_a24 to up to 1.16 g L−1 [7]. Although extensive research has been directed toward optimization studies, yet there is scarce knowledge on the performance and viability of large-scale photosynthetic PHB production lines.