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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
Regions of DNA that encode proteins are first transcribed into messenger RNA and then translated into protein. By examining the DNA sequence alone, we can determine the putative sequence of amino acids that will appear in the final protein. During translation, codons of three nucleotides determine which amino acid will be added next in the growing protein chain. On mRNA start codon is usually AUG, while the stop codons are UAA, UAG, and UGA. The open reading frame (ORF) is that portion of a DNA segment which will putatively code for a protein; it begins with a start codon and ends with a stop codon. Once a gene has been sequenced, it is important to determine the correct open reading frame. Every region of DNA has six possible reading frames, three in each direction because a codon consists of three nucleotides. The reading frame that is used determines which amino acids will be encoded by a gene. Typically, only one reading frame is used in translating a gene (in eukaryotes), and this is often the longest open reading frame. Once the open reading frame is known, the DNA sequence can be translated into its corresponding amino acid sequence.
Glossary of scientific and technical terms in bioengineering and biological engineering
Published in Megh R. Goyal, Scientific and Technical Terms in Bioengineering and Biological Engineering, 2018
Open reading frame (ORF) is a sequence of nucleotides in a DNA molecule that has the potential to encode a peptide or protein: it starts with a start triplet (ATG), is followed by a string of triplets each of which encodes an amino acid, and ends with a stop triplet (TAA, TAG or TGA). This term is often used when, after the sequence of a DNA fragment has been determined, the function of the encoded protein is not known. The existence of open reading frames is usually inferred from the DNA (rather than the RNA) sequence.
Molecular Biological Approaches for the Improvement of Biofuels Production
Published in Debabrata Das, Jhansi L. Varanasi, Fundamentals of Biofuel Production Processes, 2019
Debabrata Das, Jhansi L. Varanasi
Like in gene deletion, gene function also can be manipulated by overexpression of specific genes. Gene expression is the result of transcription (DNA to mRNA) and translation (mRNA to proteins) processes that occur continuously and simultaneously inside cells. By the application of DNA recombination and gene transfer technologies, it is possible to introduce a gene of interest in the genome of host organisms (Figure 5.1). Different gene expression constructs such as cDNAs (complimentary DNAs) and ORFs (open reading frames) are used for the expression of a protein of interest to enable a targeted function (Prelich 2012). The common host organisms used for overexpression studies include bacteria and yeasts. When genes are overexpressed, the amount of encoded protein or other gene product is substantially increased which leads to alteration in the function of the gene. The method of gene overexpression include several steps: (1) preparation of a plasmid construct with the gene of interest comprising a strong promoter and transcriptional enhancer elements, (2) transformation and integration of plasmid constructs into the genome of the host cells, (3) selection and characterization of stably transformed cell lines, and (4) multiplication and production of mutant transgenic strains that overexpress the protein of interest. Many overexpression studies have reported enhancement of the yields of different biofuels. The increase in lipid production was observed by heterologous expression of AtWRI1 transcription factor in Nannochloropsis salina (Kang et al. 2017). Similarly, Tian and others (2017) showed that a strain of Clostridium thermocellum expressing the pdc gene from A. pasteurianus and the adhA gene from T. saccharolyticum was able to produce high titers of ethanol using cellulose as substrate. It is observed that gene overexpression and gene knockout strategies can be performed simultaneously to obtain high yields of a product. For example, over-expression of transcriptional activator Fhl a, elimination of uptake hydrogenase, and knockout of the formate transporter in E. coli BW25113 led to a 4.6-fold increase in hydrogen production from glucose (Maeda et al. 2007). These studies indicate that gene editing techniques (gene knockout or gene overexpression) can coherently improve biofuel production.
Biotransformation of chromium (VI) by Bacillus sp. isolated from chromate contaminated landfill site
Published in Chemistry and Ecology, 2020
Md. Ekramul Karim, Shamima Akhtar Sharmin, Md. Moniruzzaman, Zeenath Fardous, Keshob Chandra Das, Subrata Banik, Md. Salimullah
In the present study, an attempt was made to amplify a common chromate resistance determinant (CRD), chromate reductase (ChrR) gene, known to involve in the transformation of Cr(VI) to Cr (III) (Figure S1). A partial amplified ChrR gene fragment (270 bp) was sequenced and searched for potential protein-encoding segments by the NCBI open reading frame (ORF) finder tool . A putative conserved domain of 89 amino acid was found and BLASTp analysis of the translated amino acid sequence has showed a high degree of homology with various chromate reductases available in the GenBank. Further, multiple sequence alignment and phylogenetic analysis of the closely related sequences showed 100% identity (89/89) and clustering to the NADH-dependent oxidoreductase of Escherichia coli (Accession No. HAJ1743128.1) (Figure 4). Several studies implicated the role of ChrR gene in bacterial chromate resistance and reduction mechanisms [45, 54, 55]. Aguilar et al. [56] demonstrated that the ChrR gene plays an important role in the functioning of ChrA (which encodes a putative chromate efflux protein driven by the membrane potential) in Shewanella sp. strain ANA-3; since when this gene is altered, ChrA no longer confers resistance to chromate. According to Zhu et al. [54] ChrA gene was up-regulated under Cr (VI) stress and heterologous expression analysis indicated its involvement with Cr (VI) resistance.
Increased removal of cadmium by Chlamydomonas reinhardtii modified with a synthetic gene for γ-glutamylcysteine synthetase
Published in International Journal of Phytoremediation, 2020
René Piña-Olavide, Luz M. T. Paz-Maldonado, M. Catalina Alfaro-De La Torre, Mariano J. García-Soto, Angélica E. Ramírez-Rodríguez, Sergio Rosales-Mendoza, Bernardo Bañuelos-Hernández, Ramón Fernando García De la-Cruz
The synthetic gene for γ-glutamylcysteine synthetase (gshA) was produced by GenScript (Piscataway, NJ), considering the sequence P0A6W9 reported in the UniProtKB database and the codon usage of C. reinharditii. The construct pAERR, having the synthetic gene gshA flanked by NcoI and PciI restriction sites to facilitate its subcloning, was generated as previously reported (Ramírez-Rodríguez et al.2019). The amplified gene gshA, of 1558 bp in length and including the oligonucleotides forward 5′-CATGGGAGGAGGTCCACCTGTATGATTCCAGA-3′(1F) and reverse 5′-ACATGTTCATGACATACAGGTGGACCTCCTCTT-3′(1R), was cloned into the vector pGEM®-T Easy. This vector and the prepared insert (gene gshA) were ligated at a 1:3 vector-to-insert ratio, with the reaction performed for 48 h at 4 °C, to generate the construct pRPO1 (Figure 1). The positive clones were selected based on restriction profiles to confirm the presence of the gene gshA, with conventional sequencing performed to confirm the preservation of the correct open reading frame.
Arsenic removal using Chlamydomonas reinhardtii modified with the gene acr3 and enhancement of its performance by decreasing phosphate in the growing media
Published in International Journal of Phytoremediation, 2019
Angélica E. Ramírez-Rodríguez, Bernardo Bañuelos-Hernández, Mariano J. García-Soto, Dania G. Govea-Alonso, Sergio Rosales-Mendoza, M. Catalina Alfaro de la Torre, Elizabeth Monreal-Escalante, Luz M. T. Paz-Maldonado
The acr3 gene sequence was obtained from the PubMed database (GenBank: ADP20954.1) and synthesized by GenScript (Piscataway, NJ), taking into account the codon usage of C. reinharditii. The synthetic gene was flanked by NcoI and BstEII restriction sites to facilitate its subcloning. The gene, of 1 kb in length, was released from the cloning vector pUC57. The pCAMBIA-1304 binary vector was digested with the same enzymes with the subsequent release of a 2 kb fragment corresponding to reporter genes. This pCAMBIA-1304 vector possesses a kanamycin-resistance cassette for bacteria and a hygromycin-resistance cassette for screening transformed plants. The prepared insert (gene acr3) and vector were ligated at a 3:1 insert-to-vector ratio to generate the pARR1 vector, which drives the expression of acr3 under the constitutive 35SCaMV promoter. We selected positive clones based on restriction profiles to verify the presence of the gene acr3, with conventional sequencing confirming the preservation of the correct open reading frame. Our group sequenced this synthetic gene and registered it in GenBank under the accession number MF375641.