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Results for “microbial engineering”

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At least 487 records · Page 27Linked to original sources
PubMed1986

Promoter-probe vectors for the analysis of divergently arranged promoters.

A series of plasmid-based promoter-probe vectors has been constructed which are particularly useful for the analysis of divergent control regions. Each vector contains a pair of divergently oriented indicator genes whose expression can be monitored over a wide range by simple assay methods. These genes are separated by different polylinkers. Specifically, the beta-galactosidase gene (lacZ) was employed in combination with either the galactokinase gene (galK) or the alkaline phosphatase gene (phoA). In all cases translational stop codons are present in all three reading frames upstream from the initiation codon. The vectors permit direct detection of promoters--independent of insert orientation--on indicator plates after transformation. Using this vector system, we further characterized the divergent tet control regions of transposon Tn10 and plasmid pBR322.

Alkaline Phosphatase↗
PubMed2026

Mechanisms linking the gut microbiota to colorectal cancer development and progression.

Colorectal cancer remains a leading cause of global cancer mortality, with a concerning rise in early-onset cases driven by complex interactions between environmental exposures, lifestyle factors, and host genetics. Mounting evidence indicates that gut microbiota dysbiosis critically modulates this oncogenic process, acting as an active participant rather than a passive bystander. This review systematically synthesizes the dichotomous roles of the intestinal microbiome in colorectal tumorigenesis through the conceptual framework of the driver-passenger model. We discuss how early initiating driver bacteria, such as Polyketide synthase-positive Escherichia coli and enterotoxigenic Bacteroides fragilis, compromise mucosal barriers, induce chronic mucosal inflammation, and inflict direct genomic instability. As the local tumor microenvironment undergoes profound metabolic remodeling, opportunistic passenger pathogens, notably Fusobacterium nucleatum, become enriched, further promoting cellular proliferation and facilitating tumor immune evasion. Conversely, protective commensals, exemplified by Clostridium butyricum and Streptococcus thermophilus, exert robust tumor-suppressive effects through multifaceted mechanisms. These beneficial microbes actively antagonize malignant progression by redirecting tumor metabolic fluxes toward oxidative stress, orchestrating deep epigenetic reprogramming, and degrading core oncoproteins to reverse chemoresistance. Transitioning from fundamental mechanisms to clinical application, we evaluate a comprehensive spectrum of microbiota-targeted interventions, encompassing non-invasive diagnostic biomarkers, fecal microbiota transplantation, engineered bacteria, phage therapy, and postbiotics. Finally, we critically address the formidable translational challenges associated with microbial heterogeneity, long-term safety, and regulatory standardization, aiming to provide a balanced perspective on integrating microbiome-based strategies into next-generation precision oncology for colorectal cancer.

Humans↗
PubMedDate not supplied

Microbial impacts to the near-field environment geochemistry: a model for estimating microbial communities in repository drifts at Yucca Mountain.

Geochemical and microbiological modeling was performed to evaluate the potential quantities and impact of microorganisms on the geochemistry of the area adjacent to and within nuclear waste packages in the proposed repository drifts at Yucca Mountain, Nevada. The microbial growth results from the introduction of water, ground support, and waste package materials into the deep unsaturated rock. The simulations, which spanned 1 million years, were accomplished using a newly developed computer code, Microbial Impacts to the Near-Field Environment Geochemistry (MING). MING uses environmental thresholds for limiting microbial growth to temperatures below 120 degrees C and above relative humidities of 90% in repository drifts. Once these thresholds are met, MING expands upon a mass balance and thermodynamic approach proposed by McKinley et al. [FEMS Microbiol. Rev. 20 (1997) 545] by using kinetic rates to supply constituents from design materials and constituent fluxes including solubilized rock components into the drift to perform two separate mass balance calculations as a function of time. The first (nutrient limit) assesses the available nutrients (C, N, P and S) and calculates how many microorganisms can be produced based on a microorganism stoichiometry of C(160)(H(280)O(80))N(30)P(2)S. The second (energy limit) calculates the energy available from optimally combined redox couples for the temperature and pH at that time. This optimization maximizes those reactions that produce >15 kJ/mol (limit on useable energy) using an iterative linear optimization technique. The final available energy value is converted to microbial mass at a rate of 1 kg of biomass (dry weight) for every 64 MJ of energy. These two values (nutrient limit and energy limit) are then compared and the smaller value represents the number of microorganisms that can be produced over a specified time. MING can also be adapted to investigate other problems of interest as the model can be used in saturated and unsaturated environments and in laboratory situations to establish microbial growth limitations. Other projected uses include investigations of contaminated locations where monitored natural attenuation or engineered bioremediation could be employed.

Carbon↗
PubMed2005

Synthetic biology: engineering Escherichia coli to see light.

We have designed a bacterial system that is switched between different states by red light. The system consists of a synthetic sensor kinase that allows a lawn of bacteria to function as a biological film, such that the projection of a pattern of light on to the bacteria produces a high-definition (about 100 megapixels per square inch), two-dimensional chemical image. This spatial control of bacterial gene expression could be used to 'print' complex biological materials, for example, and to investigate signalling pathways through precise spatial and temporal control of their phosphorylation steps.

Agar↗
PubMed1988

An alternative approach to somatic cell gene therapy.

Mouse primary skin fibroblasts were infected with a recombinant retrovirus containing human factor IX cDNA. Bulk infected cells capable of synthesizing and secreting biologically active human factor IX protein were embedded in collagen, and the implant was grafted under the epidermis. Sera from the transplanted mice contain human factor IX protein for at least 10-12 days. Loss of immunoreactive human factor IX protein in the mouse serum is not due to graft rejection. Instead, the mouse serum contains anti-human factor IX antibodies, which react with the protein. We suggest that retroviral-infected primary skin fibroblasts offer an alternative approach to somatic cell gene therapy.

Animals↗
PubMed1984

Expression of galactokinase as a fusion protein in Escherichia coli and Saccharomyces cerevisiae.

Plasmids are described that allow fusions between the Escherichia coli galK gene (coding for galactokinase) and any gene of interest. An example is given in which a galK gene, lacking the normal initiator methionine codon, is fused to various segments of the 5' end of the tetR gene of pBR322. The resulting plasmids complemented an E. coli galK mutant, and galactokinase activity was retained despite the addition of up to 250 foreign amino acids to the amino-terminus of the galactokinase polypeptide. In a second experiment, the galK gene was fused to the LEU2 gene of Saccharomyces cerevisiae. The resulting plasmid was able to complement a yeast GAL1-mutant and galactokinase synthesis in yeast was controlled, via the LEU2 regulatory system, by the levels of leucine and threonine in the growth medium. The galK fusion plasmids should facilitate analysis of the control systems of a wide variety of genes in different organisms.

Base Sequence↗
PubMed1999

Constructs for insertional mutagenesis, transcriptional signal localization and gene regulation studies in root nodule and other bacteria.

Cassettes have been developed that contain an antibiotic resistance marker with and without a promoterless gusA reporter gene. The nptII (encoding kanamycin resistance) or aacCI (encoding gentamicin resistance) genes were equipped with the tac promoter (Ptac) and the trpA terminator (TtrpA) and then cloned between NotI sites to construct the CAS-Nm (Ptac-nptII-TtrpA) and CAS-Gm (Ptac/PaacCI-aacCI-TtrpA) cassettes. The markers were also cloned downstream to a modified promoterless Escherichia coli gusA gene (containing TGA stop codons in all three reading frames prior to its RBS and start codon) to construct the CAS-GNm (gusA-Ptac-nptII-TtrpA) or CAS-GGm (gusA-Ptac/PaacCI-aacCI-TtrpA) cassettes. Cassettes containing the promoterless gusA create type I fusions with a target DNA sequence to detect transcriptional activity. The promoterless gusA gene has also been cloned into a broad-host-range IncP1 plasmid. This construct will enable transcriptional activity to be monitored in different genetic backgrounds. Each cassette was cloned as a NotI fragment into the NotI site of a pUT derivative to construct four minitransposons. The mTn5-Nm (containing Ptac-nptII-TtrpA) and mTn5-Gm (containing Ptac/PaacCI-aacCI-TtrpA) minitransposons have been constructed specifically for insertional inactivation studies. The minitransposons mTn5-GNm (containing gusA-Ptac-nptII-TtrpA) and mTn5-GGm (containing gusA-Ptac/PaacCI-aacCI-TtrpA) can be used for transcription signal localization or insertional inactivation. The TAC-31R and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-Nm, CAS-Gm, mTn5-Nm and mTn5-Gm. The WIL3 and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-GNm, CAS-GGm, mTn5-GNm and mTn5-GGm. The specific application of these constructs to generate acid- or nodule-inducible fusions is presented. The new constructs provide useful tools for insertional mutagenesis, transcriptional signal localization and gene regulation studies in the root nodule bacteria and possibly other gram-negative bacteria.

Base Sequence↗
PubMed1994

An optimization study of a pH-inducible promoter system for high-level recombinant protein production in Escherichia coli.

The unique properties exhibited by the pH-inducible promoter system are clearly demonstrated by the plasmid construct, pSM552-545C-. Step changes of pH substantially increase the expression of beta-galactosidase. Very high expression, a level of around 40% of total cellular protein, can be achieved with superbroth. The high level of induction in rich media, typical of those commonly used to achieve high cell density, suggests the system is versatile enough to be adapted to many specific situations. The variable degree of induction by pH within the range of 8.0 and 5.5 makes possible a degree of expression control not easily accomplished with the existing systems. By precise monitoring of induction pH, a "fine tuning" of foreign gene expression and growth rate to optimum levels is possible. The effect of several operating parameters on recombinant protein production are evaluated. Our results show that operating environments play an extremely important role in achieving high recombinant protein expression levels in a dense culture. Under suboptimal conditions, as are shown in this study, only moderately high levels can be obtained. Even for suboptimal cases, an expression level of about 10 to 15% of total cellular protein while achieving an optical density higher than 25 is routinely obtained. Our results also show that a proper balance between cell growth and recombinant protein synthesis processes are critical in maintaining high expression levels in a dense culture. Any imbalance will most likely lead to more cell growth and poorer protein productivity. We have also demonstrated that reactor operating temperature can be a useful parameter to fine-tune this balance, resulting in significantly improved results.

Biotechnology↗
PubMed1976

A restriction endonuclease analysis of the bacterial plasmid controlling the ecoRI restriction and modification of DNA.

Genetic analyses of DNA restriction and modification mechanisms have been encumbered by the inability to rigorously select for mutant phenotypes associated with these systems. The application of restriction endonucleases has now proved to be a successful approach to the genetic analyses of small genomes that are recalcitrant to the more standard genetic techniques. Restriction endonucleases EcoRI and HindIII were used to analyze the structure of the plasmid genome responsible for the EcoRI restriction endonuclease and modification methylase. This plasmid in the original clinical isolate of Escherichia coli appears to be identical to the ColE 1 plasmid except for a 1.95 kilobase pair segment which contains these genes. A preliminary restriction map of this plasmid is presented.

Base Sequence↗
PubMed1994

Genetically engineered synthesis of natural products.

The feasibility of multi-step, one-flask total synthesis of natural products is demonstrated by cloning and overexpression of the corresponding biosynthetic enzymes from plant and microbial sources. As many as eight or nine enzymes can be recombined in vitro to reach an advanced intermediate in the vitamin B12 pathway.

Amino Acid Sequence↗
PubMed2006

Expanded thermodynamic model for microbial true yield prediction.

Thermodynamic methods to predict true yield and stoichiometry of bacterial reactions have been widely used in biotechnology and environmental engineering. However, yield predictions are often inaccurate for certain simple organic compounds. This work evaluates an existing method and identifies the cause of prediction errors for compounds with low degree of reductance of carbon. For these compounds, carbon, not energy or reducing equivalents, constrains growth. Existing thermodynamically-based models do not account for the potential of carbon-limited growth. The improved method described here consists of four balances: carbon balance, nitrogen balance, electron balance, and energy balance. Two efficiency terms, K1 and K2 are defined and estimated from a priori analysis. The results show that K1 and K2 are nearly the same in value so that only one coefficient, K = 0.41 is used in the modified model. Comparisons with observed yields show that use of the new model and parameters results in significantly improved yield estimation based on inclusion of the carbon balance. The average estimation error is less than 6% for the data set presented.

Bacteria↗
PubMed1997

Recombinant plasmid mobilization between E. coli strains in seven sterile microcosms.

Transfer by mobilization of a pBR derivative recombinant plasmid lacking transfer functions (oriT+, tra-, mob-) from one E. coli K12 strain to another was investigated in seven sterile microcosms corresponding to different environments. These microcosms were chosen as representative of environments that genetically engineered microorganisms (GEMOs) encounter after accidental release, namely attached biomass in aquatic environments (biofilm), soil, seawater, freshwater, wastewater, mouse gut, and mussel gut, GEMOs survived in the same way as the host strains in all microcosms. Recombinant DNA mobilization occurred in the mouse gut, in sterile soil, and in biofilm. The plasmid transfer rates principally reflected the environmental conditions encountered in each microcosm.

Animals↗
PubMed1998

The use of affinity adsorbents in expanded bed adsorption.

The potential for the use of affinity ligands in expanded bed adsorption (EBA) procedures is reviewed. The use of affinity ligands in EBA may improve its use in direct recovery operations, as the enhanced selectivity of the adsorbent permits selective capture of the target from complex feedstocks and high degrees of purification. The properties of ligands suitable for use in EBA processes are identified and illustrated with examples. In addition to its use in the recovery of soluble products, such as proteins and nucleic acids, from particulate feedstocks, EBA can also be used to recover particulate entities, such as cells and packaged DNA (viruses and phages), from feedstocks. Affinity ligands coupled to appropriate chosen support materials will be required for such processes in order to achieve the necessary selectivity for the required particulate entity. The latter point is illustrated by the use of proteinaceous ligands immobilized to perfluorocarbon emulsions to achieve separations of microbial cells.

Adsorption↗
PubMed1993

Microbial retention of mercury from waste streams in a laboratory column containing merA gene bacteria.

The microorganisms used for the mercury retention experiments were natural isolates and genetically engineered bacteria. All mercury-resistant strains contained the merA gene. Column experiments with these strains were carried out by immobilizing them on different support materials. To obtain kinetic data of the reductase activity for whole cells and the crude extract, batch experiments were carried out under different conditions.

Aeromonas↗
PubMed2003

AHMII: Agent to Help Microbial Information Integration.

Reproducibility is the key to science. Therefore, the fidelity of organisms, particularly type strains, used in molecular biology must be authenticated before embarking on a series of experiments. In the case of microbes the authentic strain can be obtained from a culture collection. However, tracking down a culture collection that possesses the strain can be an arduous task. With this in mind we have developed a one-stop search engine for bacteria, fungi, yeasts and cell lines that indicates which collections carry the strain of interest. The search agent is named Agent to Help Microbial Information Integration (AHMII) and the URL address is http://www.wdcm.org/AHMII/ahmii.html.

Bacteria↗
PubMed2006

Phage display in the study of infectious diseases.

Microbial infections are dependent on the panoply of interactions between pathogen and host and identifying the molecular basis of such interactions is necessary to understand and control infection. Phage display is a simple functional genomic methodology for screening and identifying protein-ligand interactions and is widely used in epitope mapping, antibody engineering and screening for receptor agonists or antagonists. Phage display is also used widely in various forms, including the use of fragment libraries of whole microbial genomes, to identify peptide-ligand and protein-ligand interactions that are of importance in infection. In particular, this technique has proved successful in identifying microbial adhesins that are vital for colonization.

Adhesins, Bacterial↗