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How to identify genes in Ralstonia that synthesize PHB …

, and (2003)Poly(3-hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli harbouring Streptomyces aureofaciens PHB biosynthesis genes: Effect of various carbon and nitrogen sources. Microbiological Research, 158. pp. 19-27. ISSN 0944-5013

Recombinant Escherichia coli (ATCC:PTA–1579) harbouring poly(3-hydroxybutyrate) (PHB) synthesising genes from Streptomyces aureofaciens NRRL 2209 accumulates PHB. Effects of different carbon and nitrogen sources on PHB accumulation by recombinant E. coli were studied. Among the carbon sources used glycerol, glucose, palm oil and ethanol supported PHB accumulation. No PHB accumulated in recombinant cells when sucrose or molasses were used as carbon source. Yeast extract, peptone, a combination of yeast extract and peptone, and corn steep liquor were used as nitrogen sources. The maximum PHB accumulation (60% of cell dry weight) was measured after 48 h of cell growth at 37°C in a medium with glycerol as the sole carbon source, and yeast extract and peptone as nitrogen sources. Scanning electron microscopy of the PHB granules isolated from recombinant E. coli revealed these to be spherical in shape with a diameter ranging from 0.11 to 0.35 μm with the mean value of 0.23 ± 0.06 μm.

Genes responsible for the synthesis of poly(3-hydroxybutyrate) (PHB) in Azotobacter sp

How to identify genes in Ralstonia that synthesize PHB and ..

N2 - We have increased the gene dosage of the poly(3-hydroxybutyrate) biosynthesis operon in Ralstonia eutrophia (formerly Alcaligenes eutrophus) to test whether PHB synthesis rates may be increased by recombinant methods. The native R. eutropha phb CAB operon was inserted into the broad-host-range vector pKT230. This PHB operon-containing plasmid, and a control plasmid containing the identical broad-host-range replicon but not the PHB genes, were transferred to R. eutropha H16. Analysis of whole-cell lysates indicated that the strain harboring the operon-containing plasmid possessed β-ketothiolase and acetoacetyl-CoA reductase specific activities that were 6.0 and 6.2 times elevated, respectively, as compared to the control strain with a single operon. After growth on fructose, PHB synthesis rates were sharply dependent on the type of carbon source offered during the PHB accumulation phase under nitrogen limitation. In the case of the strain harboring the control plasmid, and in comparison to fructose as carbon source, PHB accumulation was 2.15, 2.83, and 2.60 times faster when resuspended in nitrogen-free medium with lactate, acetate, or 3-hydroxybutyrate, respectively. The strain harboring the PHB operon-containing plasmid synthesized PHB at a lower specific rate in each case. During exponential growth on fructose, the strain harboring the control plasmid was again more efficient at forming PHB. These results suggest that increasing the intracellular concentration of PHB precursors may be a superior alternative to raising the levels of PHB enzymes for enhancing PHB productivity in R. eutropha.

The genes that we manipulated and the choice of our pathway was informed by the types of organic compounds that are able to serve as substrates for PHB synthesis. We searched through literature and found several articles that analyzed solid human waste. We discovered that human fecal waste contains volatile fatty acids (short-chain fatty acids such as acetic acid, propionic acid, and butyric acid), long chain fatty acids, and glucose which can be used as substrates for PHB production (Rose ., 2015). We wanted our system to be able to make use of a wide range of carbon sources and transform them into our desired product, PHB. Therefore, we decided to manipulate pathways and genes that use these substrates to synthesize PHB. (Hiroe et al., 2012; Balck & DiRusso, 1994; DAvid et al., 2008; Tsuge et al., 2011)VFAs and long-chain fatty acids can be broken down through the fatty acid β-oxidation pathway to synthesize PHB (Davis et al., 2008). We manipulated this pathway by transforming the bacteria with the genes and . Glucose can be broken down into acetyl-CoA, which is then converted to PHB by genes involved in PHB synthesis: , and (Hiroe et al. 2012). This pathway can also indirectly use VFAs as its source for PHB production.

Cloning of the Alcaligenes eutrophus genes for synthesis of ..

Under suboptimal growth conditions and like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly-βhydroxybutyrate (PHB). The two genes, bdhA (3-hydroxybutyrate dehydrogenase) and acsA2 (acetoacetyl-CoA synthetase), which are considered to be involved in the PHB degradation pathway in Azospirillum brasilense strain Sp7, were identified, cloned, and sequenced. Additionally, the expression of the bacterial genes phbA (βketothiolase) and phbC (PHB synthase), which are involved in PHB biosynthesis and the expression of the acsA2 gene, were studied using GUS fusions. Our results indicate that these genes are constitutively expressed in Azospirillum brasilense Sp7 and that the Ntr, PII and PZ nitrogen regulatory systems, which have been shown to be involved in the regulation of PHB synthesis, do not affect the expression of these genes. Expression of these genes is also shown to occur during association of A. brasilense with wheat roots.

AB - We have increased the gene dosage of the poly(3-hydroxybutyrate) biosynthesis operon in Ralstonia eutrophia (formerly Alcaligenes eutrophus) to test whether PHB synthesis rates may be increased by recombinant methods. The native R. eutropha phb CAB operon was inserted into the broad-host-range vector pKT230. This PHB operon-containing plasmid, and a control plasmid containing the identical broad-host-range replicon but not the PHB genes, were transferred to R. eutropha H16. Analysis of whole-cell lysates indicated that the strain harboring the operon-containing plasmid possessed β-ketothiolase and acetoacetyl-CoA reductase specific activities that were 6.0 and 6.2 times elevated, respectively, as compared to the control strain with a single operon. After growth on fructose, PHB synthesis rates were sharply dependent on the type of carbon source offered during the PHB accumulation phase under nitrogen limitation. In the case of the strain harboring the control plasmid, and in comparison to fructose as carbon source, PHB accumulation was 2.15, 2.83, and 2.60 times faster when resuspended in nitrogen-free medium with lactate, acetate, or 3-hydroxybutyrate, respectively. The strain harboring the PHB operon-containing plasmid synthesized PHB at a lower specific rate in each case. During exponential growth on fructose, the strain harboring the control plasmid was again more efficient at forming PHB. These results suggest that increasing the intracellular concentration of PHB precursors may be a superior alternative to raising the levels of PHB enzymes for enhancing PHB productivity in R. eutropha.

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pha/phb genes once PHB synthesis is ..


Genetic Engineering - BiologyMad

Davis, R., Anilkumar, P.K., Chandrashekar, A. & Shamala, T.R. (2008). Biosynthesis of polyhydroxyalkanoates co-polymer in using genes from and Antonie Van Leeuwenhoek. 94: 207-216

PSEN1 Gene - GeneCards | PSN1 Protein | PSN1 Antibody

The overarching goal for the synthesis component of the project was to produce poly-3-hydroxybutyrate (PHB) by utilizing the nutrients present in human waste. To accomplish efficiently convert organic feedstocks into PHB, we genetically engineered bacteria to produce PHB by manipulating two metabolic systems within as follows:

AKT1 Gene - GeneCards | AKT1 Protein | AKT1 Antibody

Today biotechnology can be used in a variety of ways such as in an industrial setting where they use it to create enzymes to synthesize chemicals, in an environmental setting where they use it for waste and pollution prevention and lastly it can be used in medical applications such as in pharmaceuticals, genetic engineering, DNA fingerprinting and in lastly it can be used in stem cell therapy (Keener and Hoban et al., 2014)....

Industrial applications of microbial lipases - ScienceDirect

Please note that the result at this point was already scaled up (please refer to "secretion" from the table above. The value 1.5x1021 was chosen as the number of cells because this was the order of magnitude that would give the reasonable amount of enzyme for degradation. Other parameters such as those involved in gene expression were the closest we could get them to and the enzyme kinetics data were taken from the our own enzyme assay experiment. Therefore, we were left with the number of cells and the unknown secretion rate to modify. After several runs of simulation, we decided to increase the number of cells to 1.5x1021, instead of the 1.5x1012 assumed in the synthesis model.

RECENT PUBLICATIONS - Toyama Prefectural University

This study represents the first report on the synthesis of Nm and fabrication of Nm-PHB nanocomposite film which show strong protective effect against multidrug resistant Staphyloccoccus aureus.

Free biotechnology Essays and Papers - 123HelpMe

Therefore, we substituted the original pathway with our synthetic pathway. Moreover, we added our gene expression models for the enzymes into the metabolic model as well. In terms of the consistency of the model, we changed the units of our synthetic pathway into micro molar and seconds.

Alginate and PHB synthesis in A. vinelandii - Biology Online

Bioplastics are an alternative substitute for petrochemical synthetic plastics. Polyhydroxybutyrate (PHB) genes are involved in bioplastic synthesis. In this study, bioplastic synthesis genes were incorporated into the genome of oil palm because this plant has a high concentration of the PHB precursor acetyl-CoA. Immature embryos (IEs) of Elaeis guineensis var Tenera were infected with Agrobacterium tumefaciens LBA4404 that contained the binary vector pJLPHB3, which encoded the phb genes, β-ketothiolase (bktB), acetoacetyl-CoA reductase (phaB) and PHA synthase (phaC) flanked by a modified CaMV35S promoter, a plastid targeting sequence and the nos terminator. GUS assay revealed that about 78-100% transient transformation frequency was obtained for calluses and 55-65% for plantlets 1 month after transformation. However, GUS assays of leaf tissue from 12-month-old plantlets showed that only 10-33% transformation frequency was obtained. The presence of the phb genes in GUS positive plantlets was confirmed using PCR and multiplex PCR analyses. Southern blot analyses verified that the phb genes were integrated in transformed leaves and calluses using the phaB probe (0.805 kb) and phaC probe (1.730 kb). Quantitative transgene expression comparison in the transformed tissues measured using real-time PCR showed that the expression levels of the phaB and phaC transgenes were 6.06- and 6.02-fold higher compared to the non-transformed oil palm.

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