China 
Africa 
Explore chapters and articles related to this topic
Produced by Recombinant Bacteria
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
The simplest construct for expression in E. coli was the high-copy-number plasmid pUCl 19 carrying the Thermus isomerase gene, under control of the inducible lacUVS-promoter. This plasmid was named pUCTXI. In plasmid pUSTXI, the isomerase gene is carried by a pBR322 derivative, pUS12, under control of the strong and inducible tac promoter. This promoter is reportedly stronger than the lacUVS promoter. In pUCTXI and pUSTXI the Thermus ribosome-binding site remains between the host ribosome-binding site and the Thermus translation start condón, GTG. Therefore, plasmid pKKTXI was constructed in which the isomerase gene was fused to the translation start codon, ATG, of the trc promoter, which is nearly identical with the tac promoter. The main differences between pKKTXI and pUSTXI are (1) removal of the 57 Thermus nucleotides between the host ribosome-binding site and the translation start; (2) change of the translation start codon from GTG to the more common ATG; (3) optimization of the codon usage for the NH2-terminal eight amino acids, in particular change of a AGG for arginine to CGT (70% usage in E. coli). In pETTXI the translation start codons ATG of the isomerase gene and the bacteriophage T7 4>10 promoter are fused together. The cf>10 promoter is specifically recognized by T7 RNA polymerase, which is much more active than E. coli RNA polymerase (e.g., chain elongation is five times faster).
Molecular Biology and Bioinformatics in Industrial Microbiology and Biotechnology
Published in Nduka Okafor, Benedict C. Okeke, Modern Industrial Microbiology and Biotechnology, 2017
Nduka Okafor, Benedict C. Okeke
The mRNA is transcribed from one strand of the DNA of the gene; it is translated at the ribosome into a polypeptide sequence. Translation is the synthesis of protein from amino acids on a template of messenger RNA in association with a ribosome. The bases on mRNA code for amino acids in triplets or codons; that is three bases code for an amino acid. Sometimes, different triplet bases may code for the same amino acid. Thus, the amino acid glycine is coded for by four different codons: GGU, GGC, GGA, and GGG. There are 64 different codons; three of these UAA, UAG, and UGA are stop codons and end the process of translation. The remaining 61 codons code for the amino acids in proteins (Table 3.1). Translation of the message generally begins at AUG, which also codes for methionine. For AUG to act as a start codon, it must be preceded by a ribosome binding site. If that is not the case, it simply codes for methionine.
Recombinant DNA technology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
The rDNA technology has been gaining importance over the past few years, especially now that genetic diseases have become more prevalent and the agricultural area is reduced. rDNA technology has a great impact on growing better crops (drought- and heat-resistant crops), making of recombinant vaccines (such as for Hepatitis B), prevention and cure of sickle cell anemia and cystic fibrosis, production of clotting factors, insulin and recombinant pharmaceuticals, plants that produce their own insecticides, and germ line and somatic gene therapy. The rDNA works when the host cell expresses protein from the recombinant genes. The host will only produce significant amounts of recombinant protein if expression factors are added. Protein expression depends upon the gene being surrounded by a collection of signals that provide instructions for the transcription and translation of the gene by the cell. These signals include the promoter, the ribosome-binding site, and the terminator. Expression vectors in which the foreign DNA is inserted contain these signals. Signals are species specific. In the case of E. coil, these signals must be E. coil signals, as E. coil is unlikely to understand the signals of human promoters and terminators. Problems are encountered if the gene contains introns or contains signals that act as terminators to a bacterial host. This results in premature termination, and the recombinant protein may not be processed correctly, may be folded incorrectly, or may even be degraded. Production of recombinant proteins in eukaryotic systems generally takes place in yeast and filamentous fungi. The use of animal cells is a challenging approach because many need a solid support surface, unlike bacteria, and have complex growth needs. However, some proteins are too complex to be produced in bacterium, so eukaryotic cells must be used.
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.