scholarly journals Standardization of Inducer-Activated Broad Host Range Expression Modules: Debugging and Refactoring an Alkane-Responsive AlkS/PalkB Device

2021 ◽  
Author(s):  
Alejandro Arce-Rodríguez ◽  
Ilaria Benedetti ◽  
Jose Manuel Borrero-de Acuña ◽  
Rafael Silva-Rocha ◽  
Víctor de Lorenzo
Keyword(s):  
Dna Sequences ◽  
Recombinant Dna ◽  
Nitrogen Sources ◽  
Broad Host Range ◽  
Recombinant Dna Technology ◽  
Carbon And Nitrogen ◽  
Expression Of Genes ◽  
Heterologous Expression Systems ◽  
Composition Standard

Abstract Although inducible heterologous expression systems have been available since the birth of recombinant DNA technology, the diversity of genetic devices and their coming together in the corresponding vectors often result in a lack of reproducibility and interoperability. In an effort to increase predictability of expression of genes of interest in a variety of possible bacterial hosts we propose a composition standard for debugging and reassembling all regulatory parts that participate in the performance of such devices. As a case study we addressed the n-octane and dicyclopropyl ketone (DCPK)-inducible PalkB promoter of the alkane biodegradation pOCT plasmid of Pseudomonas putida. The standardized expression module included an edited alkS transcription factor divergently expressed and separated from PalkB by a synthetic buffer sgement. The DNA sequence of the alkS gene was modified to alleviate the catabolite repression exerted by several carbon and nitrogen sources through the Crc/Hfq complex of some hosts. The PalkB promoter and the alkS variants were then formatted as SEVA (Standard European Vector Architecture) cargoes and their activity in P. putida quantified with GFP and luminiscent reporters. Despite considerable editing of the DNA sequences involved, the thereby refactored module basically kept the functioning parameters of the original configuration. The same qualities were inspected when the system was passed to Escherichia coli and P. aeruginosa. We argue that application of the compositional standard thereby implemented in the AlkS/PalkB module to other promoter/regulator pairs will enable more complex genetic programming in non-model bacteria.

scholarly journals Standardization of inducer-activated broad host range expression modules: Debugging and refactoring an alkane-responsive AlkS/PalkB device

2020 ◽  
Author(s):  
Alejandro Arce-Rodríguez ◽  
Ilaria Benedetti ◽  
Rafael Silva-Rocha ◽  
Víctor de Lorenzo
Keyword(s):  
Recombinant Dna ◽  
Regulatory Gene ◽  
Nitrogen Sources ◽  
Broad Host Range ◽  
Recombinant Dna Technology ◽  
Carbon And Nitrogen ◽  
Synthetic Dna ◽  
Expression Of Genes ◽  
Conditional Expression ◽  
Heterologous Expression Systems

ABSTRACTAlthough inducible heterologous expression systems have been available since the birth of recombinant DNA technology, the diversity of devices and genetic architectures of the corresponding vectors have often resulted in a lack of reproducibility and interoperability. In an effort to increase predictability of expression of genes of interest in a variety of possible bacterial hosts we propose a composition standard for debugging and reassembling all regulatory parts that participate in the performance of such devices. As a case study we address the n-octane and dicyclopropyl ketone (DCPK)-inducible PalkB promoter of the alkane biodegradation pOCT plasmid of Pseudomonas putida. The standardized expression module consisted of an edited alkS regulatory gene that is divergently expressed and separated of PalkB by a synthetic DNA buffer sequence. The native DNA sequence of the structural alkS gene was modified to alleviate the catabolite repression exerted by some carbon and nitrogen sources through the Crc/Hfq complex of some hosts. The PalkB promoter along with the alkS variants were then formatted as SEVA (Standard European Vector Architecture) cargoes and their activity parameters in P. putida determined with GFP and luminiscent reporters. The thereby refactored system showed improvements in various features desirable in conditional expression modules: inducibility, capacity, noise reduction and on/off ratio. When applied to other promoter/regulator pairs, the compositional standard thereby implemented in the AlkS/PalkB module will enable more complex genetic programming in non-model bacteria.

Progress in the molecular cytogenetics of man

1988 ◽  
Vol 319 (1194) ◽  
pp. 239-248 ◽  
Keyword(s):  
Dna Sequences ◽  
Recombinant Dna ◽  
Molecular Cytogenetics ◽  
Chromosome Analysis ◽  
Parental Origin ◽  
Human Chromosomes ◽  
Recombinant Dna Technology ◽  
Chromosome Deletions ◽  
Dna Technology

Recombinant DNA technology has contributed greatly to the precision of chromosome analysis in man. Breakpoints of chromosome deletions and rearrangements may be defined on a chromosome map whose landmarks are the loci of DNA sequences rather than Giemsa bands. Flow cytogenetics allows the extent of chromosome duplications and deletions to be measured more precisely than has hitherto been possible. DNA probes can reveal hidden translocations through the application of in situ hybridization, and may be used as markers to determine the parental origin of non-disjunction. It is evident that a study of the pathology of human chromosomes now requires the combined skills of recombinant DNA and cytology.

scholarly journals Recombinant DNA technology and DNA sequencing

2019 ◽  
Vol 63 (4) ◽  
pp. 457-468 ◽  
Author(s):  
Mark A. J. Roberts
Keyword(s):  
Dna Sequencing ◽  
Human Genome ◽  
Dna Sequences ◽  
Human Genome Project ◽  
Recombinant Dna ◽  
Genome Project ◽  
Recombinant Dna Technology ◽  
Dna Technology ◽  
The Human Genome Project

Abstract DNA present in all our cells acts as a template by which cells are built. The human genome project, reading the code of the DNA within our cells, completed in 2003, is undoubtedly one of the great achievements of modern bioscience. Our ability to achieve this and to further understand and manipulate DNA has been tightly linked to our understanding of the bacterial and viral world. Outside of the science, the ability to understand and manipulate this code has far-reaching implications for society. In this article, we explore some of the basic techniques that enable us to read, copy and manipulate DNA sequences alongside a brief consideration of some of the implications for society.

scholarly journals Bone morphogenetic protein 2: heterologous expression and potential in bone regeneration

2021 ◽  
Vol 43 (114) ◽  
Author(s):  
Andrea Mesa Restrepo ◽  
Juan Fernando Alzate ◽  
Edwin Bairon Patiño Gonzalez
Keyword(s):  
Heterologous Expression ◽  
Bone Morphogenetic Protein ◽  
Recombinant Dna ◽  
Redox System ◽  
Bone Morphogenetic Protein 2 ◽  
Recombinant Dna Technology ◽  
Morphogenetic Protein ◽  
Alkaline Phosphate ◽  
Heterologous Expression Systems

Currently, bone morphogenetic protein 2 (BMP-2) is one of the two osteoinductive growth factors used in medical devices to promote bone formation. Typically, this protein is bought from commercial houses at high rates and in small quantities that are not enough to cover clinical needs. Because of this, it has been proposed that research centers use their own heterologous expression systems to have a constant supply of BMP-2. The aim of this study was to standardize the heterologous expression of BMP-2 and evaluate its osteoinductive activity in vitro. Our procedure for expression and purification was based on recombinant DNA technology using the plasmid pET-28 and IPTG as inductor. After extracting the protein from inclusion bodies, folding it and modifying it via a redox system, we observed via electrophoresis a 26 kDa dimer. We evaluated its osteoinductive activity in myoblastic C2C12 by quantifying enzymatically the activity of alkaline phosphate (ALP) and staining mineralization nodules. ALP activity is proportional to BMP-2 concentration, increasing 90% at 3 µg/mL. These cells form calcium nodules, mineralizing 50% of the area.

Genetically engineered reagents

1990 ◽  
Vol 333 (1628) ◽  
pp. 93-98
Keyword(s):  
Biological Samples ◽  
Recombinant Dna ◽  
Genetically Engineered ◽  
Site Directed Mutagenesis ◽  
Cloning And Expression ◽  
Specific Gene ◽  
Recombinant Dna Technology ◽  
Expression Of Genes ◽  
Protein Molecules ◽  
Dna Technology

The development of recombinant DNA technology has led to the wide availability of reagents for the analysis of trace quantities of macromolecules and small chemical agents. The cloning and expression of genes coding for the synthesis of proteins and enzymes has meant that previously scarce protein molecules have now become available as both standards for their own measurement in biological samples and as reagents for the determination and measurement of other molecules. In addition the advent of recombinant DNA technology has led to the development and advancement of valuable ‘spin off’ capabilities such as (i) site directed mutagenesis providing the ability to specifically alter the amino acid sequence and structure of protein molecules at the level of the gene, (ii) the synthesis of DNA probes to provide for both the isolation and detection of specific gene sequences in biological samples and (iii) gene amplification employing techniques such as the polymerase chain reaction to amplify and provide multiple hundred to thousands of copies of the specific gene sequence.

CRISPR/Cas9 genome editing technology applications in biological and biomedical fields

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Gene Therapy ◽  
Genome Editing ◽  
Dna Sequences ◽  
Recombinant Dna ◽  
Genetic Disorders ◽  
Specific Gene ◽  
Recombinant Dna Technology ◽  
P Gene ◽  
A Genome ◽  
Biomedical Fields

Gene therapy is a way of mending or replacing a gene in an undesirable or non-functional cell. Although used in both animals and plants, gene therapy is most usually linked with humans. Because there are so many genetic disorders caused by genetic abnormalities or unwanted gene expression, gene therapy is promising to treat and even cure many diseases. The scientific and pharmaceutical sectors are becoming interested in gene therapy.CRISPR was initially detected in prokaryotic organisms, bacteria and archaea genomes. Although nucleotide sequences are regularly discovered in many bacteria and archaea, the scientific community has not realized its importance for over a decade. People used these diverse DNA sequences as a diagnostic for genotyping and therefore considered them as a distinctive feature for each particular microbe. Scientists are beginning to comprehend that the CRISPR/Cas system is a prokaryotic defense system's adaptive immunity to viruses, due to the discovery of CRISPR-associated protein (Cas) and the use of recombinant DNA technology. This recently discovered CRISPR/Cas system was swiftly developed as a tool for editing a specific gene in a genome. Since 2012, CRISPR/Cas9 genome editing technology has been quickly researched and applied in several biological and biomedical fields. For various basic and practical research reasons, as well as biotechnological applications in agriculture and healthcare, CRISPR/Cas9 technology has altered and improved greatly over the past five years. Base editor invention and prime editing technology by fusing a Cas endonuclease with other functional enzymes, such as base converter enzymes, is one of several milestones in this fast progress.

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