College of Engineering

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College of Engineering Biomedical Engineering Biotechnology Doctoral Degrees - UMass Lowell 2013
College of Engineering Biomedical Engineering Biotechnology Doctoral Degrees at UMass Lowell 2013 Graduate Commencement Ceremonies on May 18, 2013 Learn ...

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College of Engineering

Directed evolution of an E. coli inner membrane transporter for improved efflux of biofuel molecules

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Background:
The depletion of fossil fuels and the rising need to meet global energy demands have led to a growing interest in microbial biofuel synthesis, particularly in Escherichia coli, due to its tractable characteristics. Besides engineering more efficient metabolic pathways for synthesizing biofuels, efforts to improve production yield by engineering efflux systems to overcome toxicity problems is also crucial. This study aims to enhance hydrocarbon efflux capability in E. coli by engineering a native inner membrane transporter, AcrB, using the directed evolution approach.
Results:
We developed a selection platform based on competitive growth using a toxic substrate surrogate, which allowed rapid selection of AcrB variants showing enhanced efflux of linear and cyclic fuel molecule candidates, n-octane and alpha-pinene. Two mutants exhibiting increased efflux efficiency for n-octane and alpha-pinene by up to 47% and 400%, respectively, were isolated. Single-site mutants based on the mutations found in the isolated variants were synthesized and the amino acid substitutions N189H, T678S, Q737L and M844L were identified to have conferred improvement in efflux efficiency. The locations of beneficial mutations in AcrB suggest their contributions in widening the substrate channel, altering the dynamics of substrate efflux and promoting the assembly of AcrB with the outer membrane channel protein TolC for more efficient substrate export. It is interesting to note that three of the four beneficial mutations were located relatively distant from the known substrate channels, thus exemplifying the advantage of directed evolution over rational design.
Conclusions:
Using directed evolution, we have isolated AcrB mutants with improved efflux efficiency for n-octane and alpha-pinene. The utilization of such optimized native efflux pumps will increase productivity of biofuels synthesis and alleviate toxicity and difficulties in production scale-up in current microbial platforms.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/81

RMUTSV. ARCHITECTURAL THESIS 2012. RESEARCH AND DEVELOPMENT BIOTECHNOLOGY CENTER THAKSIN UNIVERSITY – Video

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RMUTSV. ARCHITECTURAL THESIS 2012. RESEARCH AND DEVELOPMENT BIOTECHNOLOGY CENTER THAKSIN UNIVERSITY
RMUTSV. ARCHITECTURAL THESIS2012. PROJECT : RESEARCH AND DEVELOPMENT BIOTECHNOLOGY CENTER THAKSIN UNIVERSITY PHATTHALUNG CAMPUS BY : Mr.TEERAPONG KHAMPUANG A...

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RMUTSV. ARCHITECTURAL THESIS 2012. RESEARCH AND DEVELOPMENT BIOTECHNOLOGY CENTER THAKSIN UNIVERSITY - Video

The development and use of an ELISA-based method to follow the distribution of cellulase monocomponents during the hydrolysis of pretreated corn stover

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Background:
It is widely recognised that fast, effective hydrolysis of pretreated lignocellulosic substrates requires the synergistic action of multiple types of hydrolytic and some non-hydrolytic proteins. However, due to the complexity of the enzyme mixture, the enzymes interaction with and interference from the substrate and a lack of specific methods to follow the distribution of individual enzymes during hydrolysis, most of enzyme-substrate interaction studies have used purified enzymes and pure cellulose model substrates. As the enzymes present in a typical "cellulase mixture" need to work cooperatively to achieve effective hydrolysis, the action of one enzyme is likely to influence the behaviour of others. The action of the enzymes will be further influenced by the nature of the lignocellulosic substrate. Therefore, it would be beneficial if a method could be developed that allowed us to follow some of the individual enzymes present in a cellulase mixture during hydrolysis of more commercially realistic biomass substrates.
Results:
A high throughput immunoassay that could quantitatively and specifically follow individual cellulase enzymes during hydrolysis was developed. Using monoclonal and polyclonal antibodies (MAb and PAb, respectively), a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed to specifically quantify cellulase enzymes from Trichoderma reesei: cellobiohydrolase I (Cel7A), cellobiohydrolase II (Cel6A), and endoglucanase I (Cel7B). The interference from substrate materials present in lignocellulosic supernatants could be minimized by dilution.
Conclusion:
A double-antibody sandwich ELISA was able to detect and quantify individual enzymes when present in cellulase mixtures. The assay was sensitive over a range of relatively low enzyme concentration (0 -- 1 mug/ml), provided the enzymes were first pH adjusted and heat treated to increase their antigenicity. The immunoassay was employed to quantitatively monitor the adsorption of cellulase monocomponents, Cel7A, Cel6A, and Cel7B, that were present in both Celluclast and Accellerase 1000, during the hydrolysis of steam-pretreated corn stover (SPCS). All three enzymes exhibited different individual adsorption profiles. The specific and quantitative adsorption profiles observed with the ELISA method were in agreement with earlier work where more labour intensive enzyme assay techniques were used.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/80

Cellulase activity mapping of Trichoderma reesei cultivated in sugar mixtures under fed-batch conditions

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Background:
On-site cellulase production using locally available lignocellulosic biomass (LCB) is essential for cost-effective production of 2nd-generation biofuels. Cellulolytic enzymes (cellulases and hemicellulases) must be produced in fed-batch mode in order to obtain high productivity and yield. To date, the impact of the sugar composition of LCB hydrolysates on cellulolytic enzyme secretion has not been thoroughly investigated in industrial conditions.
Results:
The effect of sugar mixtures (glucose, xylose, inducer) on the secretion of cellulolytic enzymes by a glucose-derepressed and cellulase-hyperproducing mutant strain of Trichoderma reesei (strain CL847) was studied using a small-scale protocol representative of the industrial conditions. Since production of cellulolytic enzymes is inducible by either lactose or cellobiose, two parallel mixture designs were performed separately. No significant difference between inducers was observed on cellulase secretion performance, probably because a common induction mechanism occurred under carbon flux limitation. The characteristics of the enzymatic cocktails did not correlate with productivity, but instead were rather dependent on the substrate composition. Increasing xylose content in the feed had the strongest impact. It decreased by 2-fold cellulase, endoglucanase, and cellobiohydrolase activities and by 4-fold beta-glucosidase activity. In contrast, xylanase activity was increased 6-fold. Accordingly, simultaneous high beta-glucosidase and xylanase activities in the enzymatic cocktails seemed to be incompatible. The variations in enzymatic activity were modelled and validated with four fed-batch cultures performed in bioreactors. The overall enzyme production was maintained at its highest level when substituting up to 75% of the inducer with non-inducing sugars.
Conclusions:
The sugar substrate composition strongly influenced the composition of the cellulolytic cocktail secreted by T. reesei in fed-batch mode. Modelling can be used to predict cellulolytic activity based on the sugar composition of the culture-feeding solution, or to fine tune the substrate composition in order to produce a desired enzymatic cocktail.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/79

VIU – International Master Degree in Biotechnology of Human Assisted Reproduction and Embryology – Video

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VIU - International Master Degree in Biotechnology of Human Assisted Reproduction and Embryology
http://www.viu.es/master-human-reproduction-embryology/ This Master provides students with a highly advanced theoretical understanding of human reproductive biology; embryology; fertility...

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VIU - International Master Degree in Biotechnology of Human Assisted Reproduction and Embryology - Video

Mining for hemicellulases in the fungus-growing termite Pseudacanthotermes militaris using functional metagenomics

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Background:
The metagenomic analysis of gut microbiomes has emerged as a powerful strategy for the identification of biomass-degrading enzymes, which will be no doubt useful for the development of advanced biorefining processes. In the present study, we have performed a functional metagenomic analysis on comb and gut microbiomes associated with the fungus-growing termite, Pseudacanthotermes militaris.
Results:
Using whole termite abdomens and fungal-comb material respectively, two fosmid-based metagenomic libraries were created and screened for the presence of xylan-degrading enzymes. This revealed 101 positive clones, corresponding to an extremely high global hit rate of 0.49%. Many clones displayed either beta-d-xylosidase (EC 3.2.1.37) or alpha-l-arabinofuranosidase (EC 3.2.1.55) activity, while others displayed the ability to degrade AZCL-xylan or AZCL-beta-(1,3)-beta-(1,4)-glucan. Using secondary screening it was possible to pinpoint clones of interest that were used to prepare fosmid DNA. Sequencing of fosmid DNA generated 1.46 Mbp of sequence data, and bioinformatics analysis revealed 63 sequences encoding putative carbohydrate-active enzymes, with many of these forming parts of sequence clusters, probably having carbohydrate degradation and metabolic functions. Taxonomic assignment of the different sequences revealed that Firmicutes and Bacteroidetes were predominant phyla in the gut sample, while microbial diversity in the comb sample resembled that of typical soil samples. Cloning and expression in E. coli of six enzyme candidates identified in the libraries provided access to individual enzyme activities, which all proved to be coherent with the primary and secondary functional screens.
Conclusions:
This study shows that the gut microbiome of P. militaris possesses the potential to degrade biomass components, such as arabinoxylans and arabinans. Moreover, the data presented suggests that prokaryotic microorganisms present in the comb could also play a part in the degradation of biomass within the termite mound, although further investigation will be needed to clarify the complex synergies that might exist between the different microbiomes that constitute the termitosphere of fungus-growing termites. This study exemplifies the power of functional metagenomics for the discovery of biomass-active enzymes and has provided a collection of potentially interesting biocatalysts for further study.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/78

Ruckus over FDA’s approval to food from cloned animals

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The Food and Drug Administration is facing criticism over its recent preliminary approval to food from cloned animals as a consumer group has charged the agency for using flawed analysis. According to the Center for Food Safety, the FDA could not find studies on milk or meat from clones and whether they’re safe and the agency relied on studies done on cloned animals and whether they appeared healthy. Claiming that the conclusions drawn by the FDA was based on ‘scant data from few peer-reviewed studies’, Charles Margulis, a spokesman for Center for Food Safety, said: There isn’t the science to show that these foods are safe. I think the agency was heavily influenced by the biotechnology industry. Though FDA was tight lipped, Val Giddings, a scientist who consults with biotechnology companies, has come forward in FDA’s defence. According to Giddings an exhaustive amount of peer-reviewed data was the base of the conclusion. Giddings said: There’s not a single shred of data to suggest that food derived from clones or their offspring is in any way unsafe. All of what FDA has done here has been completely transparent. The FDA found that food from clones and food from conventional livestock has no virtual difference between and therefore special labels for cloned food would be necessary. Center for Food Safety might be in the process of waging a war against the FDA but Dean Foods Co. of Dallas has already decided to go against the idea of cloned food. Nations biggest milk company has decided it would not sell milk from cloned cows. The company’s decision was influenced by various surveys suggesting the dislike for dairy products from clones by Americans. Source.

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Bdelloid rotifers evolving over the past forty million years without sex

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We were up till now aware of the fact that sexual reproduction was the only means of bringing life into this world but a group of microscopic organisms seems to have broken this fact. A recent study has found out that since the past forty million years Bdelloid rotifers were evolving without sex. These aquatic animals thrive in wet areas and are asexual. In this case the Bdelloid rotifers produce eggs which are genetic clones of the mother and above all there is no male species, just females out there doing their job. That sounds amazing!! We are all aware of the fact that asexual animals cannot evolve and mutate over a period of time but study of the fossil records of bdelloid rotifers has stated that their existence can be traced back to forty million years. Up till now it was thought that sexual reproduction was important for spitting into divergent species but this has shocked everybody and also left a question to be answered as to how these species have been able to diverge without the addition of any genetic material. Via cbc

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Secrecy surrounding genetically engineered grapes field tests can have serious repercussions

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UC Davis and Cornell University have the approval for testing genetically engineered grapes in California. In this case no application or environmental assessments were undertaken for the permits and there were just notifications given by the institutes. As far as the field tests are concerned there seems to be a veil of secrecy surrounding them therefore grape growers are not aware regarding the measures which need to be taken for protecting their vineyards from genetic contamination which could dent their image and even cause huge losses if the customers shun genetically engineered products. USDA was even criticized for not paying attention towards the field trials being undertaken and the U.S. Inspector General report said: USDA lacks basic information about the field test sites it approves and is responsible for monitoring, including where and how the crops are being grown, and what becomes of them at the end of the field test. It was only last month when a federal judge ruling stated that USDA cannot give approval for new GE field trials without environmental assessments but this wont be applicable to the grape field tests which have been already given permission. Such secrecy is expected to cause huge problems in the future for GE foods and if proper study and transparency is not ensured then genetically modified food will have a tough time ahead. Via napavalleyregister

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South Korea again gives a go ahead to use of human eggs in cloning research

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South Korea is playing a risky game as it has given the permission for using human eggs in cloning research despite a high level scandal in their country which involved one of their top scientists admitting to his involvement in doctored research work. Hwang Woo-suk was the scientist who had claimed that he had cloned human embryos and extracted stem cells from them but it was found out that all his claims were false. What raised eyes were when eggs required for research were donated by a female scientist in his team and this questioned the ethics of such practice. This shameful incident caused Hwang Woo-suk to resign from his post at the Seoul National University and is now facing trial for misappropriation of government funds. In order to get over the shameful act the government has again given a go ahead to use of human eggs in cloning but this time with an act of caution and under a new set of guidelines has asked that researchers should only use eggs which are to be destroyed after fertility treatments or from other legal ways and a prior license would have to be obtained from the government for undertaking research. It seems this time South Korea wants to take no chances. Via theage

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