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Escherichia coli
Published in Ovide Arino, David E. Axelrod, Marek Kimmel, Mathematical population dynamics, 2020
Joseph M. Mahaffy, Judith W. Zyskind
The deterministic part of the models begins with a computation of the concentration of the consensus sequence of the DnaA binding site, TTAT(C/A)CA (C/A)A, along the chromosome away from oriC. These sites act as a reserve for the active DnaA · ATP and are assumed to be uniformly distributed along the chromosome. The sites are added at a constant rate to each elongating chromosome throughout the simulation. The time for a chromosome to replicate is called the C period for a cell and is approximately constant for the cell generation times used in the simulations (Bremmer and Dennis, 1987; Helmstetter, 1987). The total number of gene sites, GS, with the consensus sequence satisfies GS(t+Δt)=GS(t)+AC⋅kgsΔt
Industrial Chemicals: Enzymatic Transformation by Recombinant Microbes
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
The DTA gene was subcloned into pBR322 to construct the plasmid pDT320. The DTA activity of the E. coli transformant harboring pDT320 was less than that of X. oryzae IAM 1657, although the DTA gene was carried on the high-copy-number plasmid (Table 2). The putative promoter sequence was less similar to the consensus sequence of the E. coli promoter. These indicate that its own promoter of the DTA gene from X. oryzae is less efficient in the E. coli cell. Two 12-bp repeat sequences were observed in the region upstream from the DTA gene. This long repeat partly overlaps with the putative -35 and -10 promoter sequences, although the exact function is unknown (Fig. 6). The replication origin of the ColEl plasmid can function in the Xanthomonas cells as efficiently as in the E. coli cells [53]. Because the growth of Xanthomonas cells was relatively insensitive to ampicillin, resistance to this antibiotic could not be used as a selectable marker in this organism. To make possible the selection by chloramphenicol to which Xanthomonas cells are sensitive, the DTA gene was subcloned into the vector pBR328. The resultant plasmid, pDT648, was introduced into the X. citri IFO 3311. The promoter of X. oryzae IAM 1657 was able to function in X. citri IFO 3311 efficiently. The activity of X. citri transformant was almost 20 times higher than that of X. oryzae IAM 1657 (see Table 2).
Biophysical and Biochemical Characterization of Peptide, Protein, and Bioconjugate Products
Published in Sandeep Nema, John D. Ludwig, Parenteral Medications, 2019
Tapan K. Das, James A. Carroll
Glycoproteins can be either N-linked or O-linked, depending on the type of covalent modification of the glycan to the protein. The type of glycosylation is dependent on both the protein sequence and the expression system used for production. Glycosylation may commonly occur for proteins expressed in mammalian or yeast expression systems, but is not observed for proteins expressed in bacterial systems. N-linked glycosylation occurs at asparagine residues in the consensus sequence of Asn-Xxx-Ser or Asn-Xxx-Thr, where Xxx is any amino acid except proline. While the presence of this motif does not guarantee glycosylation, it makes N-linked glycosylation a predictable attribute. The amino acid sequence can be easily scanned for this sequence motif in order to determine if N-linked glycosylation is a possibility for a given biotherapeutic protein. Analysis of N-linked glycosylation, therefore, begins with an assessment of the site occupancy levels of any possible N-linked glycosylation sites in molecule, referred to as the macroheterogeneity. This can be accomplished using analytical methods which can distinguish size variants, such as electrophoretic or chromatographic separations, or mass spectrometry. For glycoproteins with multiple glycosylation sites, macroheterogeneity can lead to complex mixtures. For example, the therapeutic glycoprotein interferon gamma has two sequence motifs for N-linked glycosylation. Therefore, there are four theoretical forms based on occupancy alone: unoccupied, two different singly occupied forms, and one fully occupied form [48].
Enzymatic biodegradation, kinetic study, and detoxification of Reactive Red-195 by Halomonas meridiana isolated from Marine Sediments of Andaman Sea, India
Published in Environmental Technology, 2023
Purbasha Saha, Sonal Madliya, Anmol Khare, Ikshita Subudhi, Kokati Venkata Bhaskara Rao
The potentially effective isolate was further identified by the molecular biology technique of 16S rRNA sequencing. Genomic DNA of the potentially effective bacterium was isolated enzymatically using the standard phenol/chloroform method [26]. The isolated 16S rDNA of the bacterium was amplified with the forward primer 27 F and the reverse primer 1492 S using polymerase chain reaction (PCR) (Bio-Rad T100 Thermal Cycler). The PCR conditions were: initial denaturation at 95°C for 5 min; followed by 30 cycles of denaturation at 95°C for 30 sec, annealing at 56°C for 1 min, and extension at 72°C for 1 min; and a final reaction at 72°C for 10 min. The resulting PCR amplicons were purified and sequenced using Thermo-Fisher Sanger Sequencing kit in Applied Biosystems 3730 × l Genetic Analyser. A consensus sequence of the 16S rDNA gene was generated in the aligner software (BioEdit). The basic local alignment search tool (BLAST) was then used to compare the generated consensus sequence with other existing sequences deposited in National Centre for Biotechnology Information (NCBI). Closely related sequences along with the consensus sequence of the potentially effective isolate were aligned using MUSCLE, and a phylogenetic tree was generated using the software MEGA 7.