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Results for “strain engineering”

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At least 433 records · Page 24Linked to original sources

PubMed1996

Effects of strain distribution in the intervertebral discs on the progression of ossification of the posterior longitudinal ligaments.

STUDY DESIGN: Strain distribution in the intervertebral discs was evaluated biomechanically using an engineering true strain calculation formula. OBJECTIVES: This study was performed to clarify the involvement of dynamic factors in the progression of ossification of the posterior longitudinal ligament. SUMMARY OF BACKGROUND DATA: In patients with ossification of the posterior longitudinal ligament of the cervical spine, ossification frequently progresses after laminectomy. This suggests the involvement of dynamic factors in the progression of ossification. However, these factors have not yet been clarified. METHODS: The analysis was performed on 101 patients with ossification of the posterior longitudinal ligament by employing dynamic lateral x-ray films of the cervical spine. The x-ray films were digitized and used as computer data for calculating the strain distribution. X-ray films were obtained again 5 years later, and the strain distribution and the presence or absence of progression of ossification were evaluated. RESULTS: The progression of ossification of the posterior longitudinal ligament was highly correlated with abnormal strain distribution in the intervertebral discs. Progression of ossification was frequently observed in areas having disc distortion in tension and extension on the posterior longitudinal ligament. CONCLUSIONS: In this study, the area of progression of ossification corresponded to the area showing uneven strain distribution and resultant concentration of dynamic stress. These results suggest an important role for dynamic factors in the progression of ossification of the posterior longitudinal ligament.

Adult↗

PubMed1995

Salmonella strains and mammalian cells genetically engineered for expression of sulfotransferases.

Rat and human sulfotransferases (STs) were expressed in his- S. typhimurium strains. These new bacterial strains detected various mutagens which are difficult to recognize in traditional test systems, including benzylic alcohols derived from polycyclic aromatic hydrocarbons, hycanthone and 1'-hydroxysafrole. STs were also stably expressed in V79 Chinese hamster cells, which do not express endogenous ST and are suitable for the detection of genotoxic effects. Positive responses in these test systems were observed with various benzylic alcohols, including benzo[a]pyrene-7,8,9,10-tetrols. We demonstrate that a few reactive sulfuric acid conjugates are efficiently detected as genotoxicants only when generated directly within the indicator cell.

Animals↗

PubMed2002

Engineering of a Bacillus subtilis strain with adjustable levels of intracellular biotin for secretory production of functional streptavidin.

Streptavidin is a biotin-binding protein which has been widely used in many in vitro and in vivo applications. Because of the ease of protein recovery and availability of protease-deficient strains, the Bacillus subtilis expression-secretion system is an attractive system for streptavidin production. However, attempts to produce streptavidin using B. subtilis face the problem that cells overproducing large amounts of streptavidin suffer poor growth, presumably because of biotin deficiency. This problem cannot be solved by supplementing biotin to the culture medium, as this will saturate the biotin binding sites in streptavidin. We addressed this dilemma by engineering a B. subtilis strain (WB800BIO) which overproduces intracellular biotin. The strategy involves replacing the natural regulatory region of the B. subtilis chromosomal biotin biosynthetic operon (bioWAFDBIorf2) with an engineered one consisting of the B. subtilis groE promoter and gluconate operator. Biotin production in WB800BIO is induced by gluconate, and the level of biotin produced can be adjusted by varying the gluconate dosage. A level of gluconate was selected to allow enhanced intracellular production of biotin without getting it released into the culture medium. WB800BIO, when used as a host for streptavidin production, grows healthily in a biotin-limited medium and produces large amounts (35 to 50 mg/liter) of streptavidin, with over 80% of its biotin binding sites available for future applications.

Bacillus subtilis↗

PubMed1995

Histopathological features in the small intestine of pigs infected with F4ac+ non-enterotoxigenic or enterotoxigenic strains of Escherichia coli.

Four porcine strains of Escherichia coli were examined for their effects on the small intestine of 4-week-old weaned pigs infected orogastrically. The strains used experimentally were: strain 1467 (adhesin negative, non-toxigenic); strains 2407 and 1466 (adhesin positive, non-toxigenic), derived by genetical engineering from strain 1467 and containing a wild type plasmid and a recombinant plasmid, respectively, encoding the F4 antigen (adhesin); and strain M1823 (adhesin positive, toxigenic). In addition, 2-week-old pigs that died from natural colibacillosis associated with two strains ("Ihan 1 and 2"; adhesin positive, toxigenic) were examined. Strain M1823 and the Ihan strains produced moderate and marked lesions, respectively. Strain 1467 did not cause mucosal damage or an inflammatory response. Strains 1466 and 2407 caused a mild to moderate leucocyte (mononuclear and polymorphonuclear) infiltration in the jejunal (but not ileal) lamina propria. However, unlike strain 1466, strain 2407 did not cause damage to the small intestinal mucosa and should be further studied as a potential oral vaccine strain for post-weaning E. coli diarrhoea.

Administration, Oral↗

PubMed2003

Elastic biodegradable poly(glycolide-co-caprolactone) scaffold for tissue engineering.

Cyclic mechanical strain has been demonstrated to enhance the development and function of engineered smooth muscle (SM) tissues, and it would be necessary for the development of the elastic scaffolds if one wishes to engineer SM tissues under cyclic mechanical loading. This study reports on the development of an elastic scaffold fabricated from a biodegradable polymer. Biodegradable poly(glycolide-co-caprolactone) (PGCL) copolymer was synthesized from glycolide and epsilon-caprolactone in the presence of stannous octoate as catalyst. The copolymer was characterized by (1)H-NMR, gel permeation chromatography and differential scanning calorimetry. Scaffolds for tissue engineering applications were fabricated from PGCL copolymer using the solvent-casting and particle-leaching technique. The PGCL scaffolds produced in this fashion had open pore structures (average pore size = 250 microm) without the usual nonporous skin layer on external surfaces. Mechanical testing revealed that PGCL scaffolds were far more elastic than poly(lactic-co-glycolic acid) (PLGA) scaffolds fabricated using the same method. Tensile mechanical tests indicated that PGCL scaffolds could withstand an extension of 250% without cracking, which was much higher than withstood by PLGA scaffolds (10-15%). In addition, PGCL scaffolds achieved recoveries exceeding 96% at applied extensions of up to 230%, whereas PLGA scaffolds failed (cracked) at an applied strain of 20%. Dynamic mechanical tests showed that the permanent deformation of the PGCL scaffolds in a dry condition produced was less than 4% of the applied strain, when an elongation of 20% at a frequency of 1 Hz (1 cycle per second) was applied for 6 days. Moreover, PGCL scaffolds in a buffer solution also had permanent deformations less than 5% of the applied strain when an elongation of 10% at a frequency of 1 Hz was applied for 2 days. The usefulness of the PGCL scaffolds was demonstrated by engineering SM tissues in vivo. This study shows that the elastic PGCL scaffolds produced in this study could be used to engineer SM-containing tissues (e.g. blood vessels and bladders) in mechanically dynamic environments.

Absorbable Implants↗

PubMed2025

Exploring genetic adaptation and microbial dynamics in engineered anaerobic ecosystems via strain-level metagenomics.

Genetic heterogeneity exists within all microbial populations, with sympatric cells of the same species often exhibiting single-nucleotide variations that influence phenotypic traits, including metabolic efficiency. However, the evolutionary dynamics of these strain-level differences in response to environmental stress remain poorly understood. Here, we present a first-of-its-kind study tracking the adaptive evolution of an anaerobic, carbon-fixing microbiota under a controlled engineered ecosystem focused on carbon dioxide bioconversion into methane. Leveraging strain-resolved metagenomics with an ad hoc variant calling and phasing approach, we mapped mutation trajectories and observed that the two dominant Methanothermobacter species maintained distinct sweeping haplotypes over time, most likely due to niche-specific metabolic roles. By combining population genetic statistics and peptide reconstruction, mer and mcrB genes emerged as potential drivers of archaeal strain-level competition. These findings pave the way for targeted engineering of microbial communities to enhance bioconversion efficiency, with significant implications for sustainable energy and carbon management in anaerobic systems.

Metagenomics↗

PubMed2005

Cloning, expression, purification, and analysis of mannitol dehydrogenase gene mtlK from Lactobacillus brevis.

The commercial production of mannitol involves high-pressure hydrogenation of fructose using a nickel catalyst, a costly process. Mannitol can be produced through fermentation by microorganisms. Currently, a few Lactobacillus strains are used to develop an efficient process for mannitol bioproduction; most of the strains produce mannitol from fructose with other products. An approach toward improving this process would be to genetically engineer Lactobacillus strains to increase fructose-to-mannitol conversion with decreased production of other products. We cloned the gene mtlK encoding mannitol-2-dehydrogenase (EC 1.1.1.67) that catalyzes the conversion of fructose into mannitol from Lactobacillus brevis using genomic polymerase chain reaction. The mtlK clone contains 1328 bp of DNA sequence including a 1002-bp open reading frame that consisted of 333 amino acids with a predicted molecular mass of about 36 kDa. The functional mannitol-2-dehydrogenase was produced by overexpressing mtlK via pRSETa vector in Escherichia coli BL21pLysS on isopropyl-beta-D-thiogalactopyranoside induction. The fusion protein is able to catalyze the reduction of fructose to mannitol at pH 5.35. Similar rates of catalytic reduction were observed using either the NADH or NADPH as cofactor under in vitro assay conditions. Genetically engineered Lactobacillus plantarum TF103 carrying the mtlK gene of L. brevis indicated increased mannitol production from glucose. The evaluation of mixed sugar fermentation and mannitol production by this strain is in progress.

Cloning, Molecular↗

PubMed2004

Engineered biosynthesis of 16-membered macrolides that require methoxymalonyl-ACP precursors in Streptomyces fradiae.

Development of host microorganisms for heterologous expression of polyketide synthases (PKS) that possess the intrinsic capacity to overproduce polyketides with a broad spectrum of precursors supports the current demand for new tools to create novel chemical structures by combinatorial engineering of modular and other classes of PKS. Streptomyces fradiae is an ideal host for development of generic polyketide-overproducing strains because it contains three of the most common precursors--malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA--used by modular PKS, and is a host that is amenable to genetic manipulation. We have expanded the utility of an overproducing S. fradiae strain for engineered biosynthesis of polyketides by engineering a biosynthetic pathway for methoxymalonyl-ACP, a fourth precursor used by many 16-membered macrolide PKS. This was achieved by introducing a set of five genes, fkbG-K from Streptomyces hygroscopicus, putatively encoding the methoxymalonyl-ACP biosynthetic pathway, into the S. fradiae chromosome. Heterologous expression of the midecamycin PKS genes in this strain resulted in 1 g/l production of a midecamycin analog. These results confirm the ability to engineer unusual precursor pathways to support high levels of polyketide production, and validate the use of S. fradiae for overproduction of 16-membered macrolides derived from heterologous PKS that require a broad range of precursors.

Acyl Carrier Protein↗

PubMed1996

Recruitment and expression of toluene/trichloroethylene biodegradation genes in bacteria native to deep-subsurface sediments.

Four plasmids, each encoding a combination of either an Escherichia coli or Pseudomonas putida promoter and either toluene dioxygenase or toluene monooxygenase, were electroporated into five bacterial strains isolated from sediments found at depths of 91 to 295 m. Four of these engineered bacterial strains demonstrated both toluene and trichloroethylene degradation activities.

Bacteria↗

PubMed2026

Strategy for enhanced production of A40926B0 in Nonomuraea gerenzanensis using an efficient CRISPR/AsCas12f1 system.

The global emergence of vancomycin-resistant Gram-positive pathogens underscores the urgent need for efficient production of novel lipoglycopeptide antibiotics. Dalbavancin, a last-resort therapeutic agent, relies on its key biosynthetic precursor A40926B0, whose industrial manufacture is severely limited by the low yield of wild-type Nonomuraea gerenzanensis and inefficient genetic tools for this rare actinomycete. Here, we developed a high-efficiency CRISPR/AsCas12f1 genome editing system and applied systematic metabolic engineering to boost A40926B0 biosynthesis. First, conjugation conditions were optimized to elevate the transfer efficiency in N. gerenzanensis D11. The hypercompact AsCas12f1 nuclease showed markedly lower cytotoxicity than SpCas9 and enabled 100% gene deletion efficiency with preferred PAMs (TTTG, CTTG, GTTG). Second, we strengthened the shikimate pathway via multiple genetic strategies: overexpressing feedback-resistant DAHP synthase (aroG fbr ) and chorismate mutase/prephenate dehydrogenase (tyrA fbr ), as well as knocking out pheA. This manipulation blocks the phenylalanine synthetic branch and redirects metabolic flux toward the l-tyrosine branch. Third, we engineered the branched-chain fatty acid (BCFA) pathway via promoter replacement of bkdA2B2C2, LipAB, fabF and deletion of acdH to enhance isododecanoyl side-chain supply. The combinatorial engineering yielded strain B-13, which produced 1740 mg/L A40926B0 in shake flasks. Finally, 50-L fed-batch fermentation with continuous maltodextrin feeding further increased the titer to 1817 mg/L, the highest reported titer to date. This work establishes a robust CRISPR editing tool for N. gerenzanensis and provides valuable engineering references for precursor-oriented strain improvement targeting lipoglycopeptide antibiotics, offering insights for the industrial scale production of A40926B0.

A40926B0↗

PubMed2001

Mouse models of blood pressure regulation and hypertension.

Human essential hypertension is recognized as a multifactorial disease involving many genes, but the causative genes have not yet been identified. For many years hypertension was studied primarily in the rat, but more recently several candidate genes for hypertension have been used to produce transgenic mice for gain of function and gene-targeted mice for loss of function studies. These genetically engineered mouse strains with hypertension or hypotension are providing insights into the mechanisms of blood pressure regulation. However, genetically engineered mice are used to study one gene at a time, and another complementary approach is needed for polygenic inheritance and gene interaction. The phenotype-driven approach to hypertension studies uses the natural variation among inbred strains and crosses to find quantitative trait loci. The four mouse crosses carried out so far have found several quantitative trait loci that are concordant with hypertension loci found in rats and humans.

Animals↗

PubMed1991

Respiratory activity of alginate-encapsulated Pseudomonas fluorescens cells introduced into soil.

Alginate-entrapped cells of Pseudomonas fluorescens were introduced into soil microcosms to evaluate their respiratory activity (O2 consumption and CO2 evolution) and survival during a 14-day incubation period at 20 degrees C. Alginate-entrapped cells and cells resuspended in sterile distilled water and introduced into sterile soil exhibited relatively similar O2 consumption/CO2 evolution and survival over the 14-day period. The same treatments in non-sterile soil exhibited lower respiratory activity and a population density decrease of about 2.0 Log. cfu/g after 14 days. Alginate-entrapped bacterial cells may be a useful method for introducing genetically-engineered and non-engineered bacterial strains into the soil environment.

Alginates↗

PubMed1997

Survival of the recombinant Bacteroides thetaiotaomicron strain BTX in in vitro rumen incubations.

The survival of Bacteroides thetaiotaomicron strain BTX under rumen-simulating conditions was studied. Strain BTX is a recombinant variant of strain 5482 engineered for the production of high levels of xylanase, an enzyme important in the degradation of hemicellulose. Strain BTX was not inhibited by compounds present in rumen fluid and it grew well in media containing rumen fluid (up to 75%) or high concentrations of volatile fatty acids (total concentration, 100 mmol l-1). The ability of strain BTX to compete with other microorganisms under rumen-like conditions was studied in in vitro incubations of rumen contents. These experiments employed a consecutive batch culture (CBC) system consisting of alfalfa suspended in a rum flid buffer inoculated with blended rumen contents and maintained by transfers (10%, v/v) at 48 h intervals. CBC cultures contained a diversity of microbial morphotypes and accumulated fermentation products in rumen-like proportions. WHen added alone, the numbers of BTX cells were maintained for only a few hours, and then declined precipitously until undetectable after 48 h. If CBC cultures were also supplemented with chondroitin sulphate (a mucopolysaccharide used by Bact. thetaiotaomicron), strain BTX grew and the pattern of its population generally followed that of the total population of ruminal bacteria in these cultures. When transferred into fresh CBC cultures containing chondroitin sulphate, BTX was again able to grow and increase in numbers, but to a diminished degree. Although BTX was able to survive and maintain itself in chondroitin sulphate supplemented cultures, this was at a very low level (10(5) ml-1). The potential for manipulation of rumen function by inoculation with recombinant bacteria is discussed.

Animals↗

PubMed2002

A one-step method for genetic transformation of non-piliated Neisseria meningitidis.

A one-step method to chemically transform non-piliated Neisseria meningitidis strains previously resistant to conventional electrochemical transformation procedures has been developed. This method has been used to generate genetically engineered meningococcal strains disrupted in the structural rmpM gene encoding Rmp.

Antigens, Bacterial↗

PubMed2001

Metabolic engineering as an integrating platform for strain development.

Integration of the analytical framework and experimental tools of metabolic engineering with emerging technologies such as DNA microarrays and directed evolution stands to dramatically improve the approaches by which strain improvement and biocatalyst design are pursued in the future. Progress in genomics and applied molecular biology, together with increasing emphasis on renewable resource utilization for chemical production, has advanced metabolic engineering to the forefront of biotechnological interest.

Industrial Microbiology↗

PubMed2005

Engineering of the gut commensal bacterium Bacteroides ovatus to produce and secrete biologically active murine interleukin-2 in response to xylan.

AIMS: The aim of this work was to engineer a gut commensal bacterium, Bacteroidesovatus, to produce and secrete a biologically active cytokine in a regulated manner as a basis for novel immunotherapies for chronic gut disorders. METHODS AND RESULTS: Bacteroides ovatus was engineered to produce murine interleukin-2 (MuIL2) intracellularly in response to xylan in culture media by inserting the MuIL2 gene into the xylanase operon of the organism. A second strain was engineered to secrete MuIL2 by adding Bacteroides fragilis enterotoxin secretion signal sequence to the protein. The recombinant strains produced MuIL2 only in the presence of xylan as determined by ELISA of cell lysates and culture supernatants. The IL2-dependent cell line CTLL-2 was used to demonstrate that MuIL2 produced by both B. ovatus strains was biologically active. This activity could be blocked by an anti-IL2 neutralizing antibody. The xylan-inducible nature of this system was demonstrated by RT-PCR. CONCLUSIONS: Bacteroides ovatus was successfully engineered to produce and secrete biologically active MuIL2 in a xylan-inducible manner. SIGNIFICANCE AND IMPACT OF THE STUDY: The production and secretion of a biologically active mammalian protein by a member of the gut microflora could lead to the development of new long-term immunotherapies for inflammatory gut diseases.

Animals↗

PubMed2004

Genetically engineered animals in drug discovery and development: a maturing resource for toxicologic research.

Genetically engineered mice that either over-express a foreign gene (transgenic) or in which the activity of a specific gene has been removed ("knock-out") or replaced ("knock-in") will be used increasingly to investigate molecular mechanisms of disease, to evaluate innovative therapeutic targets, and to screen novel agents for efficacy and/or toxicity. Recent innovations of relevance to toxicologic researchers include the construction of genetically engineered mice with (1) multiple engineered genes, (2) mutations that can be induced at specific sites and times throughout life, and (3) the substitution of human genes for their mouse counterparts ("humanized" mice) to allow in vivo investigation of xenobiotic toxicity. Contemporary applications of genetically engineered mice in toxicology include basic mechanistic research exploiting newly engineered mouse lines as well as applied screening for genotoxicity and carcinogenicity using commercially available animals. Many caveats must be considered when interpreting genetically engineered mice-derived toxicity data, the chief of which will be the extent to which the model's phenotype has been fully characterized, the type and incidence of background lesions for the given mouse strain and engineered gene, and the possibility of misinterpreting the presence or absence of a phenotype due to compensatory physiologic processes that mask the outcome produced by the engineering event. Toxicity data acquired using genetically engineered mice currently supplements and in time likely will supplant those gathered using the present "gold standard" bioassays, as genetically engineered mice typically develop more lesions after a shorter latency period than do age- and strain-matched, wild-type mice.

Animals↗

PubMed2003

Use of a vaccine strain of measles virus genetically engineered to produce carcinoembryonic antigen as a novel therapeutic agent against glioblastoma multiforme.

Despite the most aggressive medical and surgical treatments, glioblastoma multiforme remains incurable with a median survival of <1 year. We investigated the antitumor potential of a novel viral agent, an attenuated strain of measles virus (MV), derived from the Edmonston vaccine lineage, genetically engineered to produce carcinoembryonic antigen (CEA). CEA production as the virus replicates can serve as a marker of viral gene expression. Infection of a variety of glioblastoma cell lines including U87, U118, and U251 at MOIs 0.1, 1, and 10 resulted in significant cytopathic effect consisting of excessive syncycial formation and massive cell death at 72-96 h from infection. terminal deoxynucleotidyltransferase-mediated nick end labeling assays demonstrated the mechanism of cell death to be predominantly apoptotic. The efficacy of this approach in vivo was examined in BALB/c nude mice by using both s.c. and intracranial orthotopic U87 tumor models. In the s.c. U87 model, mice with established xenografts were treated with a total dose of 8 x 10(7) plaque forming units of MV-CEA, administered i.v. Mice treated with UV light inactivated MV, and untreated mice with established U87 tumors were used as controls. There was statistically significant regression of s.c. tumors (P < 0.001) and prolongation of survival (P = 0.007) in MV-CEA treated animals compared with the two control groups. In the intracranial orthotopic U87 model, there was significant regression of intracranial U87 tumors treated with intratumoral administration of MV-CEA at a total dose of 1.8 x 10(6) plaque forming units as assessed by magnetic resonance image (P = 0.002), and statistically significant prolongation of survival as compared with mice that received UV-inactivated virus and untreated mice (P = 0.02). Histological examination of brains of MV-CEA-treated animals revealed complete regression of the tumor with the presence of a residual glial scar and reactive changes, mainly presence of hemosiderin-laden macrophages. In addition, CEA levels in the peripheral blood in both the s.c. and orthotopic models increased before tumor regression, indicating viral gene expression, and returned to normal when the tumors regressed. Ifnar(ko) CD46 Ge transgenic mice, susceptible to MV infection, were used to assess central nervous system toxicity of MV-CEA. Intracranial administration of MV-CEA into the caudate nucleus of Ifnar(ko) CD46 Ge did not result in clinical neurotoxicity. Pathologic examination demonstrated limited microglial infiltration surrounding the injection site. In summary, MV-CEA has potent antitumor activity against gliomas in vitro, as well as in both s.c. and orthotopic U87 animal models. Monitoring CEA levels in the serum can serve as a low-risk method of detecting viral gene expression during treatment, and could allow dose optimization and individualization of treatment.

Animals↗