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Results for “strain engineering”

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PubMed1985

Immunisation of cattle with a recombinant togavirus-vaccinia virus strain.

Genetic engineering techniques have been used to construct a vaccinia virus recombinant which contains and expresses togavirus (Sindbis) genetic information. Intradermal inoculation of this recombinant strain into calves caused a transient pock-type lesion at the site of inoculation and elicited the production of substantial levels of anti-Sindbis virus neutralising antibodies. These results suggest that recombinant vaccinia virus vaccines may have potential for use in veterinary medicine.

Animals↗

PubMed1991

Genetic engineering of Trichoderma to produce strains with novel cellulase profiles.

Genetic engineering has been used to modify the proportion of different cellulases produced by a hypercellulolytic Trichoderma reesei mutant strain. A general expression vector, pAMH110, containing the promoter and terminator sequences of the strongly expressed main cellobiohydrolase 1 (cbh1) gene was used to overexpress a cDNA coding for EGI, the major endoglucanase (1,4,beta-D-glucan glucanohydrolase, EC 3.2.1.4). An in vitro modified cbh1 cDNA, incapable of coding for active enzyme, was used to inactivate the major cellobiohydrolase (1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.91) gene. In this way, new strains producing elevated amounts of the specific endoglucanase 1 (EGI) and/or lacking the major cellobiohydrolase (CBHI) were produced, and these have been further characterized.

Base Sequence↗

PubMed2001

The role of matrix metalloproteinase-2 in the remodeling of cell-seeded vascular constructs subjected to cyclic strain.

Tissue engineering offers the opportunity to develop vascular substitutes that mimic the responsive nature of native arteries. A good blood vessel substitute should be able to remodel its matrix in response to mechanical stimulation, as imposed by the hemodynamic environment. We have developed a novel method of studying the influence of mechanical strain on the remodeling of cell-seeded collagen gel blood vessel analogs. We assessed the remodeling capacity by examining the effect of mechanical conditioning upon the expression of enzymes which remodel the extracellular matrix, called matrix metalloproteinases (MMPs), and upon the mechanical properties of the constructs. We found that subjecting collagen constructs to a 10% cyclic radial distention, over a course of 4 days, resulted in an overall increase in the production of MMP-2. Cyclic mechanical strain also stimulated enzymatic activation of latent MMP-2. We found that cyclic strain also significantly increased the mechanical strength and material modulus, as indicated by an increase in circumferential tensile properties of the constructs. These observations suggested that MMP-2-dependent remodeling affects the material properties of vascular tissue analogs. To further investigate this possible connection we examined the effects of dynamic conditioning in the presence of two nonspecific inhibitors of MMP activity. Interestingly, we found that nonspecific inhibition of MMP ablated the benefits of mechanical conditioning upon mechanical properties. Our observations suggest that a better understanding of the complex relation between mechanical stimulation and construct remodeling is key for the proper design of tissue-engineered blood vessel substitutes.

Arteries↗

PubMed2005

Bone--special problems of the craniofacial region.

PROBLEMS: The craniofacial region presents special problems for tissue engineering. First, the stresses and strains that engineered tissues will encounter are mostly unknown. Second, if tissue engineering is to be useful in ameliorating craniofacial anomalies, it will have to mimic the growth activity of the native tissues. These problems are interrelated in that bone growth responds to loading conditions. METHODS: Our work uses miniature technology to measure skull deformation during function in the miniature pig. Growth is quantified in the same animals by labeling replicating cells with bromodeoxyuridine and newly mineralized bone with fluorochromes. The mandibular condyle and the cranial sutures are both candidate areas for tissue engineering, and craniofacial periosteum is a promising graft material. RESULTS: The condyle is compressed by the reaction load at the temporomandibular joint (TMJ). Cell divisions in the perichondrium are negatively correlated with bone strain. Craniofacial sutures deform during function much more than adjacent bones, and strains can be either tensile or compressive. In contrast to expectation, functional tension is not correlated with sutural growth rate. However, functional strain does predict sutural morphology, with compressed sutures showing complex interdigitation. Periosteum shows striking differences between resorptive and appositional surfaces. The resorptive medial side of the zygomatic arch is under pressure during function. Tensile strain perpendicular to the surface is probably greater on the temporal than on the zygomatic bone, thus correlating with more rapid periosteal apposition on the temporal. CONCLUSION: Engineered implants may be more likely to succeed if their architecture suits the strain environment in which they will function.

Animals↗

PubMed2002

[Isolation and characterization of a p-nitrophenol degradation Pseudomonas sp. strain P3 and construction of a genetically engineered bacterium].

A Pseudomonas strain P3 was isolated in this work. P3 can grow with p-nitrophenol as the sole carbon and nitrogen source. Growth in media with nitrogen, P3 can degrade p-nitrophenol to accumulate nitrite in the culture media. P3 can utilize a series of aromatic compounds as sole carbon sources. Different heavy metal ions have different effects on the degradation of p-Nitrophenol by P3. Glucose had no effect on the degradation of p-nitrophenol, while trace yeast extract greatly increased the degradation rate. Methyl parathion hydrolase gene mpd was clone into P3 by conjugation and genetically engineered bacterium PM was obtained. Methyl parathion hydrolase was expressed by PM. PM could grew on methyl parathion as sole carbon source. PM could degrade methyl parathion with relatively high activity and stability.

Biodegradation, Environmental↗

PubMed2003

The relevance of large strains in functional tissue engineering of heart valves.

BACKGROUND: Exposing the developing tissue to flow and pressure in a bioreactor has been shown to enhance tissue formation in tissue-engineered heart valves. Animal studies showed excellent functionality in these valves in the pulmonary position. However, they lack the mechanical strength for implantation in the high-pressure aortic position. Improving the in vitro conditioning protocol is an important step towards the use of these valves as aortic heart valve replacements. In this study, the relevance of large strains to improve the mechanical conditioning protocol was investigated. METHODS: Using a newly developed device, engineered heart valve tissue was exposed to increasing cyclic strain in vitro. Tissue formation and mechanical properties were analyzed and compared to unstrained controls. RESULTS: Straining resulted in more pronounced and organized tissue formation with superior mechanical properties over unstrained controls. Overall tissue properties improved with increasing strain levels. CONCLUSIONS: The results demonstrate the significance of large strains in promoting tissue formation. This study may provide a methodological basis for tissue engineering of heart valves appropriate for systemic pressure applications.

Absorbable Implants↗

PubMed1993

A DNA module encoding bph genes for the degradation of polychlorinated biphenyls (PCBs).

In this report we describe the development and construction of a DNA module which encodes bph genes for the metabolism of PCBs and which is capable of stable integration into the chromosome of Gram negative bacteria. Introduction of the bph-module into Pseudomonas putida KT2442, Pseudomonas sp. strain B13 and its genetically engineered derivative B13FR1 expanded the biodegradative ability of these strains to include biphenyl and 4-chlorobiphenyl. The bph operon was stably inherited under laboratory conditions. Behavior of the genetically engineered strains was evaluated under simulated natural habitat conditions in lake sediment microcosms with respect to survival and removal of 4-chlorobiphenyl. The genetically engineered strains persisted under these conditions and were effective in degrading 4-chlorobiphenyl over a five day incubation period.

Biodegradation, Environmental↗

PubMed2005

Strain improvement by metabolic engineering: lysine production as a case study for systems biology.

A central goal of systems biology is the elucidation of cell function and physiology through the integrated use of broad based genomic and physiological data. Such systemic approaches have been employed extensively in the past, as they are a central element of metabolic flux analysis, the distribution of kinetic control in pathways, and the key differentiating characteristic of metabolic engineering. In one case study, these tools have been applied to the improvement of lysine-producing strains of Corynebacterium glutamicum. The systematic study of the physiology of this organism allowed the identification of specific metabolic targets and subsequently led to significant improvements in product yield and productivity. This case study can serve as a guide for the development of systems biology tools for the utilization of large volumes of cell- and genome-wide transcriptional and physiological data.

Corynebacterium glutamicum↗

PubMed1989

Detection in soil of a deletion in an engineered DNA sequence by using DNA probes.

Two Pseudomonas strains were engineered to contain the nptII gene and plasmid vector sequences in their chromosomes. After incubation of these strains in nonsterile soil, total bacterial DNA was isolated and analyzed by Southern blot hybridization with the nptII gene and the plasmid vector as probes. In addition to the expected bands of hybridization, a new band corresponding to the loss of vector sequences from the chromosome while retaining the nptII gene was observed for one of the strains. The more stressful conditions encountered in soil appeared to increase the frequency of loss of the vector sequences from this strain.

Chromosome Deletion↗

PubMed1991

Mutagenicity spectra in bacterial strains of airborne and engine exhaust particulate extracts.

The mutagenicity spectra of the organic extracts of both airborne particulate matter and diesel and gasoline soot particles were determined using a battery of 9 bacterial strains of different genetic specificity. The assays with crude extracts and with fractionated acidic, neutral and basic components revealed striking differences in the patterns of mutagenic responses produced by each of the complex mixtures investigated. The mutagenicity of air particulate matter was shown to depend mainly on direct-acting acidic and neutral compounds, with a lesser contribution of basic promutagens which required exogenous metabolic activation by liver S9. The assays with a diesel soot extract indicated the prevailing contribution of direct-acting acidic and neutral compounds, and suggested an important role also for nitro derivatives other than nitropyrenes. The gasoline exhaust was characterized by powerful promutagenic compounds, belonging to either the acidic, neutral or basic fractions. The implications of these results are discussed with respect to the contribution of engine exhausts to air pollution, and the possible use of mutagenicity spectra in the analysis of environmental complex mixtures.

Air Pollutants↗

PubMed1999

Recombinant polyketide synthesis in Streptomyces: engineering of improved host strains.

Efficient polyketide synthesis derived from plasmid-borne heterologous Streptomyces polyketide synthase (PKS) gene clusters necessitates a suitable host strain. Well-characterized laboratory strains such as Streptomyces coelicolor or Streptomyces lividans and their frequently used derivatives carry endogenous genes for the synthesis of actinorhodin (among other PKS genes), which might interfere with the efficient production of extrachromosomally encoded PKS proteins and the quantitative analysis of their secreted polyketide products. To circumvent this problem, a frequently used S. coelicolor derivative, designated CH999, was engineered to lack most of the actinorhodin gene cluster. However, this strain can only be transformed with methyl-free DNA. Additionally, unlike its otherwise isogenic parent CH1, CH999 exhibits low transformation efficiencies. Here, we report the construction of two S. lividans host strains, K4-114 and K4-155. With respect to the actinorhodin gene cluster, both are genotypically identical to CH999; however, both can be transformed at considerably higher frequencies and also with methylated DNA. Upon transformation with the appropriate expression vector, CH999, K4-114 and K4-155 all produce the erythromycin precursor 6-deoxyerythronolide B (6-dEB) equally well.

DNA Methylation↗

PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗

PubMed1998

Use of inducible feedback-resistant N-acetylglutamate synthetase (argA) genes for enhanced arginine biosynthesis by genetically engineered Escherichia coli K-12 strains.

The goal of this work was to construct Escherichia coli strains capable of enhanced arginine production. The arginine biosynthetic capacity of previously engineered E. coli strains with a derepressed arginine regulon was limited by the availability of endogenous ornithine (M. Tuchman, B. S. Rajagopal, M. T. McCann, and M. H. Malamy, Appl. Environ. Microbiol. 63:33-38, 1997). Ornithine biosynthesis is limited due to feedback inhibition by arginine of N-acetylglutamate synthetase (NAGS), the product of the argA gene and the first enzyme in the pathway of arginine biosynthesis in E. coli. To circumvent this inhibition, the argA genes from E. coli mutants with feedback-resistant (fbr) NAGS were cloned into plasmids that contain "arg boxes," which titrate the ArgR repressor protein, with or without the E. coli carAB genes encoding carbamyl phosphate synthetase and the argI gene for ornithine transcarbamylase. The free arginine production rates of "arg-derepressed" E. coli cells overexpressing plasmid-encoded carAB, argI, and fbr argA genes were 3- to 15-fold higher than that of an equivalent system overexpressing feedback-sensitive wild-type (wt) argA. The expression system with fbr argA produced 7- to 35-fold more arginine than a system overexpressing carAB and argI genes on a plasmid in a strain with a wt argA gene on the chromosome. The arginine biosynthetic capacity of arg-derepressed DH5 alpha strains with plasmids containing only the fbr argA gene was similar to that of cells with plasmids also containing the carAB and argI genes. Plasmids containing wt or fbr argA were stably maintained under normal growth conditions for at least 18 generations. DNA sequencing identified different point mutations in each of the fbr argA mutants, specifically H15Y, Y19C, S54N, R58H, G287S, and Q432R.

Acetyltransferases↗

PubMed2005

Metabolic engineering for synthesis of aryl carotenoids in Rhodococcus.

Rhodococcus erythropolis naturally synthesizes monocyclic carotenoids: 4-keto-gamma-carotene and gamma-carotene. The genes and the pathway for carotenoid synthesis in R. erythropolis were previously described. We heterologously expressed a beta-carotene desaturase gene (crtU) from Brevibacterium in Rhodococcus to produce aryl carotenoids such as chlorobactene. Expression of the crtU downstream of a chloramphenicol resistance gene on pRhBR171 vector showed higher activity than expression downstream of a native 1-deoxyxylulose-5-phosphate synthase gene (dxs) on pDA71 vector. Expression of the crtU in the beta-carotene ketolase (crtO) knockout Rhodococcus host produced higher purity chlorobactene than expression in the wild-type Rhodococcus host. Growth of the engineered Rhodococcus strain in eight different media showed that nutrient broth yeast extract medium supplemented with fructose gave the highest total yield of chlorobactene. This medium was used for growing the engineered Rhodococcus strain in a 10-l fermentor, and approximately 18 mg of chlorobactene was produced as the almost exclusive carotenoid by fermentation.

Biotechnology↗

PubMed2026

Deletion of the Salmonella pathogenicity island 2 gene, spiC, in attenuated Salmonella Typhimurium VNP20009 optimizes its potential for bacterial schwannoma therapy.

UNLABELLED: Recent advances in systems biology and immunotherapy have spurred the investigation of bacteria as therapeutic vehicles for cancer treatment. Currently, Bacillus Calmette-Guérin remains the only FDA-approved bacterial cancer therapy; it is a live attenuated mycobacterium that is indicated for the treatment and prophylaxis of carcinoma in situ of the urinary bladder and for the prophylaxis of primary or recurrent papillary tumors following transurethral resection. Although safety concerns have been raised, attenuated Salmonella Typhimurium strains such as VNP20009 have advanced to clinical trials targeting fast-growing human tumors. Notably, this strain induces robust immunological control of slow-growing tumors such as NF2-related schwannomatosis (NF2-SWN) in preclinical murine models. Here, we genetically characterize VNP20009 with the goal of constructing genetically defined attenuated strains that retain its promising therapeutic features while improving safety. Specifically, we investigated the contribution of the Salmonella pathogenicity island I (SPI-1) and SPI-2 type III secretion systems to antitumor efficacy and biosafety. Mutation of the SPI-1 gene sipB, a key structural component required for SPI-1 type III secretion system function, partially reduced tumor control in NF2-SWN murine schwannoma models, suggesting that bacterial invasion alone does not fully account for antitumor activity. In contrast, deletion of the SPI-2 gene spiC, a key effector required for intracellular survival, preserved robust tumor regression in NF2-SWN murine schwannoma models while improving safety and reducing systemic toxicity. To create a genetically defined and tractable platform, we generated two attenuated strains-AST101 and AST101-ΔspiC-which retain key mutations present in VNP20009 but lack ill-characterized background mutations. In the syngeneic NF2-SWN mouse schwannoma model, both strains significantly suppressed tumor growth compared to PBS. Collectively, these findings support the development of rationally engineered Salmonella Typhimurium strains with enhanced safety and preserved antitumor efficacy. IMPORTANCE: Given long-standing safety concerns surrounding the therapeutic use of live bacteria, we constructed a ΔspiC mutant of VNP20009 and demonstrated that it provides a markedly improved safety profile while retaining antitumor efficacy in NF2-related schwannomatosis mouse schwannoma models. In addition, we created two genetically defined Salmonella Typhimurium strains, AST01 and AST01-ΔspiC, which incorporate the key-targeted mutations found in VNP20009 and VNP20009-ΔspiC, respectively. These engineered strains offer a well-defined genetic background, enabling precise investigation of the bacterial traits responsible for Salmonella Typhimurium-mediated tumor control and thus further improvement of attenuated strains optimized for bacteriotherapy of neoplasms.

Salmonella typhimurium↗

PubMedDate not supplied

[Photoquenching of the bioluminescence of the genetically engineered Escherichia coli TG1 (pXen7) strain in the presence of photodithazine].

The photoquenching of the bioluminescence of the genetically engineered Escherichia coli TG1 (pXen7) strain was studied in the presence of the photosensitizer photodithazine, a glucosamine salt of chlorin e6. The photosensitized quenching of the bioluminescence was found to correlate with the colony-forming ability of the strain. The data obtained are discussed from the standpoint of using biosensor luminescent bacterial systems for the assessment of the efficiency of photosensitizers in antimicrobial photochemotherapy.

Biosensing Techniques↗

PubMed1998

Genetic engineering of an industrial strain of Saccharopolyspora erythraea for stable expression of the Vitreoscilla haemoglobin gene (vhb).

Several Actinomycetes/Streptomycetes expression vectors are described for expression of the Vitreoscilla haemoglobin gene (vhb) in an industrial erythromycin-producing strain of Saccharopolyspora erythraea. Cloning of vhb under the control of either the thiostrepton-inducible PtipA promoter or the constitutive PermE* promoter led to the production of chemically active haemoglobin (VHb) in Streptomyces lividans TK24 transformed with these constructs. However, theplasmids could not be transformed into Sac. erythraea. Transformants of Sac. erythraea and/or exconjugants were obtained using a novel Escherichia coli/Streptomyces shuttle vector comprised of vhb under the control of the PermE* promoter, the Streptomyces plasmid pIJ350 origin of replication, the thiostrepton-resistance gene (tsr) for selection, and the oriT region which is necessary for conjugal transfer. Increased plasmid stability in Sac. erythraea was obtained by construction of a vector for chromosomal integration. This vector contained the Streptomyces phage phi C31 attachment site for chromosomal integration and vhb expressed under the PmerR promoter and was stably maintained in the chromosome of Sac. erythraea. Shake-flask cultivations of the transformed Sac. erythraea strain with the chromosomally integrated vhb gene show that vhb is expressed in an active form. The corresponding amount of erythromycin produced in the vhb-expressing strain was approximately 60% higher relative to the original VHb-negative strain.

Bacterial Proteins↗

PubMed2000

Transneuronal retrograde dual viral labelling of central autonomic circuitry: possibilities and pitfalls.

Viral retrograde transneuronal labelling has become an important neuroanatomical tract-tracing tool for characterization of limbic neuronal networks. Recently, dual viral retrograde transneuronal labelling has been introduced; a method employing differential transgene expression of two genetically engineered virus strains to identify double infected cells with selective antibodies. In this way, interactions of parallel networks can be revealed. The use of this method will increase the understanding of the function of the limbic system, for example in the maintenance of metabolic homeostasis, but is associated with limitations related to the use of genetically engineered virus strains. Virulence, speed of replication and retrograde transport may be affected by the insertion or deletion of genes in the viral genome. Moreover, the rate of replication and transport can be affected by the immune system of the host and competition between the two viruses. There may be selective affinity of the virus strain for the sympathetic or parasympathetic systems. False negative results are the most important risk in dual viral labelling addressed in this review. Several control experiments are presented that can help to reduce the risk of obtaining false negative results.

Animals↗