1. Biotechnology

Category Archives: Biotechnology

Metabolic engineering of Escherichia coli for high-specificity production of isoprenol and prenol as next generation of biofuels

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Background:
The isopentenols, including isoprenol and prenol, are excellent alternative fuels. However, they are not compounds largely accumulated in natural organism. The need for the next generation of biofuels with better physical and chemical properties impels us to develop biosynthetic routes for the production of isoprenol and prenol from renewable sugar. In this study, we use the heterogenous mevalonate-dependent (MVA) isoprenoid pathway for the synthesis of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) intermediates, and then convert IPP and DMAPP to isoprenol and prenol, respectively.
Results:
A mevalonate titer of 1.7 g/L was obtained by constructing an efficient MVA upper pathway in engineered E. coli. Different phosphatases and pyrophosphatases were investigated for their abilities in hydrolyzing the IPP and DMAPP. Consequently, ADP-ribose pyrophosphatase was found to be an efficient IPP and DMAPP hydrolase. Moreover, ADP-ribose pyrophosphatase from Bacillus subtilis (BsNudF) exhibited a equivalent substrate specificity towards IPP and DMAPP, while ADP-ribose pyrophosphatase from E. coli (EcNudF) presented a high substrate preference for DMAPP. Without the expression of any phosphatases or pyrophosphatases, a background level of isopentenols was synthesized. When the endogenous pyrophosphatase genes (EcNudF and yggV) that were capable of enhancing the hydrolyzation of the IPP and DMAPP were knocked out, the background level of isopentenols was still obtained. Maybe the synthesized IPP and DMAPP were hydrolyzed by some unknown hydrolases of E. coli. Finally, 1.3 g/L single isoprenol was obtained by blocking the conversion of IPP to DMAPP and employing the BsNudF, and 0.2 g/L ~80% prenol was produced by employing the EcNudF. A maximal yield of 12% was achieved in both isoprenol and prenol producing strains.
Conclusions:
To the best of our knowledge, this is the first successful report on high-specificity production of isoprenol and prenol by microbial fermentation. Over 1.3 g/L isoprenol achieved in shake-flask experiments represents a quite encouraging titer of higher alcohols. In addition, the substrate specificities of ADP-ribose pyrophosphatases were determined and successfully applied for the high-specificity synthesis of isoprenol and prenol. Altogether, this work presents a promising strategy for high-specificity production of two excellent biofuels, isoprenol and prenol.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/57

Madison College and UW-Platteville Sign Biotechnology Articulation Agreement – Video

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Madison College and UW-Platteville Sign Biotechnology Articulation Agreement
Madison College and UW-Platteville have signed an articulation agreement that gives biotechnology students the opportunity to earn a bachelor #39;s degree and a ...

By: MadisonCollegeVideo

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Madison College and UW-Platteville Sign Biotechnology Articulation Agreement - Video

Novel 1H low field nuclear magnetic resonance applications for the field of biodiesel

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Background:
Biodiesel production has increased dramatically over the last decade, raising the need for new rapid and non-destructive analytical tools and technologies. 1H Low Field Nuclear Magnetic Resonance (LF-NMR) applications, which offer great potential to the field of biodiesel, have been developed by the Phyto Lipid Biotechnology Lab research team in the last few years.
Results:
Supervised and un-supervised chemometric tools are suggested for screening new alternative biodiesel feedstocks according to oil content and viscosity. The tools allowed assignment into viscosity groups of biodiesel-petrodiesel samples whose viscosity is unknown, and uncovered biodiesel samples that have residues of unreacted acylglycerol and/or methanol, and poorly separated and cleaned glycerol and water. In the case of composite materials, continuous distribution of exponentials, and cross-correlation methods were successfully applied to differentiate components. Continuous distributed methods were also applied to calculate the yield of the transesterification reaction, and thus monitor the progress of the common and in-situ transesterification reactions, offering a tool for optimization of reaction parameters.
Conclusions:
Comprehensive applied tools are detailed for the characterization of new alternative biodiesel resources in their whole conformation, monitoring of the biodiesel transesterification reaction, and quality evaluation of the final product, using a non-invasive and non-destructive technology that is new to the biodiesel research area. A new integrated computational-experimental approach for analysis of 1H LF-NMR relaxometry data is also presented, suggesting improved solution stability and peak resolution.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/55

Are Biodegradable Heart Stents Safe?

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A breakthrough has been achieved in the stream of medical science. An alternative to the metallic stent has been found and is called biodegradable or bio-absorbable stents.

Difference between the two

Metallic stents which are in use for a long time now, had some disadvantages. These stents helps to keep the blocked arteries open to enable the flow of oxygen and blood, but also causes retenosis, that is, it scars up vessel tissue causing the arteries to clog again. Even though drug infused metallic stents have also been used as an alternative, it still does not lower the risks of other complications.

Biodegradable stents, on the other hand causes no such complications. It opens up the blocked arteries and dissolves itself after fulfilling its task, thus, minimizing the occurrence of any complication. It is made up of poly-l-lactide, a naturally dissolving material. It is said to dissolve in a time span of 18 months to three years. Another advantage of this stent is that it does not prevent the detection of other blockages as opposed to the metallic stents which would refract the rays of the scan, making it hard for detection.

Benefits of not having a permanent stent

One of the greatest benefits of not having a permanent stent is that it allows the lumen to expand. When a permanent metallic stent is used it does not allow the lumen to grow, thus hindering remodeling even though it allows the vessel around the stent to develop.

Another benefit is they do not produce any kind of inflammatory reactions as opposed to metallic stents.

How does a biodegradable stent work?

Arteries start getting clogged up due to the accumulation of fatty matter like chlorestol on the inner wall of the arteries that are responsible for providing blood to the heart. As it advances, it reduces the width of the lumen in return diminishing the amount of blood flowing into the heart. This is when a person undergoes a chest pain known as angina.

This disease can be arrested at the initial stage with the help of medication. But a person suffers a heart attack when the precautions are not taken, or when the artery is fully obstructed. That is when the surgical procedure of angioplasty is done. In angioplasty, a balloon is introduced into the artery through a guide wire and is inflated where the blockage is located. After this the stent is introduced so that it keeps the artery open.

The biodegradable stent releases a drug called everolimus which prevents irregular tissue growth.

Researches and studies that classify biodegradable as safe

Kunhiko Kosuga, who has a MD, PhD and is also the director of cardiology at Shiga Medical Center for Adults in Moriyana City, Japan, did a research on these new stents. He and his fellow researchers studied 44 men and 6 women who had undergone angioplasty and had used biodegradable stents to open up the affected arteries. They looked for various complications like clots, deaths, and other causes. The result is as follows:

? for the deaths associated with heart diseases, the survival rate was 98%.

? for death from all causes, the survival rate was 87%.

? there was no main cardiac problems in half the patients.

? Only four patients suffered heart attacks.

? The blood vessel involved had re-narrowed in 16% of the patients, in one year after undergoing the procedure.

? there were two clots that were found within the stent. One was due to the drug-infused stent close to the biodegradable one.

Countries who welcomed biodegradable stents

Nine European countries, Middle East, parts of Latin America and parts of Asia like India, Hong Kong, Philippines and Vietnam are already using these stents. In Europe, Asia-Pacific, Canada and Latin America, over 600 patients have taken part in the trial which aspires to have 1000 patients from over 100 centres present in these counties. Even Singapore has approved of these stents from 20th December, 2012.

However, doctors are still awaiting results for the long term effects on the patients

Even though the cost for manufacturing these stents is very expensive, doctors worldwide are optimistic that they will replace metallic stents eventually.

About The Author: Alia is a writer/blogger by profession. She loves writing, travelling and reading books. She contributes to Hydroxycut

Source:
http://www.biotechblog.org/entry/biodegradable-heart-stents-safe/

Enzyme affinity to cell types in wheat straw (Triticum aestivum L.) before and after hydrothermal pretreatment

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Background:
Wheat straw used for bioethanol production varies in enzymatic digestibility according to chemical structure and composition of cell walls and tissues. In this work, the two biologically different wheat straw organs, leaves and stems, are described together with the effects of hydrothermal pretreatment on chemical composition, tissue structure, enzyme adhesion and digestion. To highlight the importance of inherent cell wall characteristics and the diverse effects of mechanical disruption and biochemical degradation, separate leaves and stems were pretreated on lab-scale and their tissue structures maintained mostly intact for image analysis. Finally, samples were enzymatically hydrolysed to correlate digestibility to chemical composition, removal of polymers, tissue composition and disruption, particle size and enzyme adhesion as a result of pretreatment and wax removal. For comparison, industrially pretreated wheat straw from Inbicon A/S was included in all the experiments.
Results:
Within the same range of pretreatment severities, industrial pretreatment resulted in most hemicellulose and epicuticular wax/cutin removal compared to lab-scale pretreated leaves and stems but also in most re-deposition of lignin on the surface. Tissues were furthermore degraded from tissues into individual cells while lab-scale pretreated samples were structurally almost intact. In both raw leaves and stems, endoglucanase and exoglucanase adhered most to parenchyma cells; after pretreatment, to epidermal cells in all the samples. Despite heavy tissue disruption, industrially pretreated samples were not as susceptible to enzymatic digestion as lab-scale pretreated leaves while lab-scale pretreated stems were the least digestible.
Conclusions:
Despite preferential enzyme adhesion to epidermal cells after hydrothermal pretreatment, our results suggest that the single most important factor determining wheat straw digestibility is the fraction of parenchyma cells rather than effective tissue disruption.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/54

"IPR in Biotechnology: A Live Session With Biological Researchers" at IIT Bombay – Video

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"IPR in Biotechnology: A Live Session With Biological Researchers" at IIT Bombay
IPR Aware World: A Social Campaign (Non-Profit Programme) Talks of following students: Gulshan Mehra Molecular Virology Lab, Hemendra Pal Singh Dhaked Molecu...

By: Sandeep Kumar Khichar

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"IPR in Biotechnology: A Live Session With Biological Researchers" at IIT Bombay - Video

The Mystery about Cyanide Taste

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What is potassium cyanide??

A chemical compound with the chemical formula KCN is commonly known as the Potassium cyanide. This is a colorless crystalline compound, highly soluble in water and is similar in appearance as that of sugar. Potassium cyanide is considered to be highly toxic in nature. Potassium cyanide is considered to be one of the most deadly compounds being discovered till date.

Production of Potassium Cyanide:

Potassium Cyanide can be produced mainly by treating hydrogen cyanide in the presence of a fifty percent of an aqueous solution of potassium hydroxide. After that the aqueous solution is either evaporated in a vacuum or by treating the form amide in the presence of potassium hydroxide. On an average per year approximately fifty thousand tons of potassium cyanide are being produced all over the world.

Use of Potassium Cyanide:

Potassium Cyanide is mainly used for the purpose of electroplating, organic synthesis of a number of chemical compounds, gold mining and so on. In accordance with the large scale use of Potassium Cyanide, it is also used in smaller scale applications like in the jewelry manufacturing industry for chemical buffing and gliding. Other than those mentioned earlier, Potassium Cyanide is also used by the entomologists as it is an excellent killing agent, and it has the unique capability of causing minimum damage to the highly fragile specimens.

Mystery about Potassium Cyanide:

Since, the time of its invention, the biggest mystery that has been surrounded with Potassium Cyanide, is about the taste of it. Due to the fact that Potassium Cyanide is exceedingly poisonous substance and can cause death of a person in seconds, the taste of it has remained a mystery or a fact yet to be known to the world. Though, many researches and tests have been conducted to find the taste of Potassium Cyanide but none of them could come up with the appropriate result.

Different views about the taste of Potassium Cyanide:

There has been number of views among the scientists about the taste of the Potassium Cyanide, some of them are as follows:

· Since on hydrolysis of KCN, the resultant compound that are formed are KOH and HCN, which are strong base and weak base respectively, Potassium Cyanide is confirmed to be basic nature. Since at room temperature HCN is a gas which evolves and the solution is expected to have more KOH, so the taste of Potassium Cyanide is assumed to be bitter.

· Some, scientists who have died while finding the taste of Potassium cyanide, could only write the alphabet “S” before dying, so it is not conclusive whether it is sour, sweet or salty in taste.

Conclusion:

Though, a huge number of scientists sacrificed their lives in order to find the taste of Potassium cyanide, but it remained a mystery for long, until and unless a goldsmith from India named MP Prasad in an attempt to commit suicide with the help of Potassium cyanide could finally reveal the taste of Potassium cyanide. As per the suicide note of him the taste of Potassium Cyanide is very much acrid, that is irritatingly harsh and sharp. This fact about the taste of Potassium cyanide is approved by the World Health Organization and is marked as an extraordinary discovery in the field of science.

Source:
http://www.biotechblog.org/entry/mystery-cyanide-taste/

Development of an electrotransformation protocol for genetic manipulation of Clostridium pasteurianum

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Background:
Reducing the production cost of, and increasing revenues from, industrial biofuels will greatly facilitate their proliferation and co-integration with fossil fuels. The cost of feedstock is the largest cost in most fermentation bioprocesses and therefore represents an important target for cost reduction. Meanwhile, the biorefinery concept advocates revenue growth through complete utilization of by-products generated during biofuel production. Taken together, the production of biofuels from low-cost crude glycerol, available in oversupply as a by-product of bioethanol production, in the form of thin stillage, and biodiesel production, embodies a remarkable opportunity to advance affordable biofuel development. However, few bacterial species possess the natural capacity to convert glycerol as a sole source of carbon and energy into value-added bioproducts. Of particular interest is the anaerobe Clostridium pasteurianum, the only microorganism known to convert glycerol alone directly into butanol, which currently holds immense promise as a high-energy biofuel and bulk chemical. Unfortunately, genetic and metabolic engineering of C. pasteurianum has been fundamentally impeded due to lack of an efficient method for deoxyribonucleic acid (DNA) transfer.
Results:
This work reports the development of an electrotransformation protocol permitting high-level DNA transfer to C. pasteurianum ATCC 6013 together with accompanying selection markers and vector components. The CpaAI restriction-modification system was found to be a major barrier to DNA delivery into C. pasteurianum which we overcame by in vivo methylation of the recognition site (5'-CGCG-3') using the M.FnuDII methyltransferase. With proper selection of the replication origin and antibiotic-resistance marker, we initially electroporated methylated DNA into C. pasteurianum at a low efficiency of 2.4 x 101 transformants mug-1 DNA by utilizing conditions common to other clostridial electroporations. Systematic investigation of various parameters involved in the cell growth, washing and pulse delivery, and outgrowth phases of the electrotransformation procedure significantly elevated the electrotransformation efficiency, up to 7.5 x 104 transformants mug-1 DNA, an increase of approximately three order of magnitude. Key factors affecting the electrotransformation efficiency include cell-wall-weakening using glycine, ethanol-mediated membrane solubilization, field strength of the electric pulse, and sucrose osmoprotection.
Conclusions:
C. pasteurianum ATCC 6013 can be electrotransformed at a high efficiency using appropriately methylated plasmid DNA. The electrotransformation method and tools reported here should promote extensive genetic manipulation and metabolic engineering of this biotechnologically important bacterium.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/50

Land-use change and greenhouse gas emissions from corn and cellulosic ethanol

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Background:
The greenhouse gas (GHG) emissions that may accompany land-use change (LUC) from increased biofuel feedstock production are a source of debate in the discussion of drawbacks and advantages of biofuels. Estimates of LUC GHG emissions focus mainly on corn ethanol and vary widely. Increasing the understanding of LUC GHG impacts associated with both corn and cellulosic ethanol will inform the on-going debate concerning their magnitudes and sources of variability.
Results:
In our study, we estimate LUC GHG emissions for ethanol from four feedstocks: corn, corn stover, switchgrass, and miscanthus. We use new computable general equilibrium (CGE) results for worldwide LUC. U.S. domestic carbon emission factors are from state-level modelling with a surrogate CENTURY model and U.S. Forest Service data. This paper investigates the effect of several key domestic lands carbon content modelling parameters on LUC GHG emissions. International carbon emission factors are from the Woods Hole Research Center. LUC GHG emissions are calculated from these LUCs and carbon content data with Argonne National Laboratory's Carbon Calculator for Land Use Change from Biofuels Production (CCLUB) model. Our results indicate that miscanthus and corn ethanol have the lowest (-10 g CO2e/MJ) and highest (7.6 g CO2e/MJ) LUC GHG emissions under base case modelling assumptions. The results for corn ethanol are lower than corresponding results from previous studies. Switchgrass ethanol base case results (2.8 g CO2e/MJ) were the most influenced by assumptions regarding converted forestlands and the fate of carbon in harvested wood products. They are greater than miscanthus LUC GHG emissions because switchgrass is a lower-yielding crop. Finally, LUC GHG emissions for corn stover are essentially negligible and insensitive to changes in model assumptions.
Conclusions:
This research provides new insight into the influence of key carbon content modelling variables on LUC GHG emissions associated with the four bioethanol pathways we examined. Our results indicate that LUC GHG emissions may have a smaller contribution to the overall biofuel life cycle than previously thought. Additionally, they highlight the need for future advances in LUC GHG emissions estimation including improvements to CGE models and aboveground and belowground carbon content data.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/51

Impact of high biomass loading on ionic liquid pretreatment

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Background:
Ionic liquid (IL) pretreatment has shown great potential as a novel pretreatment technology with high sugar yields. To improve process economics of pretreatment, higher biomass loading is desirable. The goal of this work is to establish, the impact of high biomass loading of switchgrass on IL pretreatment in terms of viscosity, cellulose crystallinity, chemical composition, saccharification kinetics, and sugar yield.
Results:
The pretreated switchgrass/IL slurries show frequency dependent shear thinning behavior. The switchgrass/IL slurries show a crossover from viscous behavior at 3 wt% to elastic behavior at 10 wt%. The relative glucan content of the recovered solid samples is observed to decrease with increasing levels of lignin and hemicelluloses with increased biomass loading. The IL pretreatment led to a transformation of cellulose crystalline structure from I to II for 3, 10, 20 and 30 wt% samples, while a mostly amorphous structure was found for 40 and 50 wt% samples.
Conclusions:
IL pretreatment effectively reduced the biomass recalcitrance at loadings as high as 50 wt%. Increased shear viscosity and a transition from 'fluid' like to 'solid' like behavior was observed with increased biomass loading. At high biomass loadings shear stress produced shear thinning behavior and a reduction in viscosity by two orders of magnitude, thereby reducing the complex viscosity to values similar to lower loadings. The rheological properties and sugar yields indicate that 10 to 50 wt% may be a reasonable and desirable target for IL pretreatment under certain operating conditions.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/52


  1. Biotechnology