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Summer 2013 Blog - Geoffrey Winegar

Geoffrey Winegar is spending ten weeks in Brasilia, Brazil working at Embrapa. 

August 20, 2013

On my last day in Brazil, I headed off to São Paulo’s Guarulhos International Airport.  At the time, I was staying with a friend in Osasco—a 40-minute drive away from the airport, according to Google Maps.  My flight was at 9:50 PM, and we left at 4:00 PM to account for traffic.  I thought this would give me plenty of time to comfortably arrive with hours to spare.  I was wrong.  After spending almost an hour stuck on a traffic-riddled bridge that was no more than a couple hundred feet long, I started to thing I would need to make arrangements for a later flight home.  Luckily, I eventually made it to the airport with just a half hour to spare—still, the drive had been made more than four hours longer than it should have been due to the congestion.

The countless cars that were idly spewing carbon dioxide may have been less startling had they all been running on ethanol, Brazil’s supposed champion of emissions-reducing alternative fuel.  Unfortunately, I have learned over the last couple months that everyone still fills up with gasoline, which remains the cheapest choice in terms of mileage per dollar because of price control by the large corporations that provide these options.  The buses provide a glimmer of hope, using a blend of biodiesel (the fuel we were optimizing at Embrapa) and petrodiesel.  Even so, the smoggy traffic-congested metropolis struck me as a reminder that scientific discoveries alone may not be enough to rectify the course we have set towards uncontrollable global warming.  Active support from political leaders, corporate powers, and the public for a green future will be necessary as well.

Indeed, it is this third group, the people, who will ultimately be the greatest driving force for protecting the environment.  I am so thankful that I have had the opportunity to live among and work with Brazilians for the past couple of months. The vigor with which they are fighting to evoke governmental changes that will provide a better future for their country has been an inspiration to me.  This same fervor exists in the lab, where a powerful sense of team unity thrusts the work forward—work that may help to preserve our beautiful planet, with its stunning variety of landscapes, wildlife, and cultures.

 

July 23, 2013

Spiders—to some, they inspire fear.  For others, they evoke an image of a vibrantly colored superhero swinging his way through downtown New York City.  To me, they are creatures worthy of utmost respect and admiration.

The webs they weave are composed of silky, modular proteins with structural motifs that are incredibly difficult to mimic.  Thus, instead of constructing the silk from the ground up artificially, we opt to transform Escherichia Coli with a recombinant silk plasmid.  Cells that are successfully transformed are isolated using a selective antibiotic (ampicillin).  A small quantity of these colonies is grown in lysogeny broth (LB) until an optical density (OD600) of .8 at 600 nm is achieved.  Then, we inoculate a larger batch of LB (usually five flasks with 1L of LB in each), and incubate once more.  We then add IPTG to induce expression of the inserted gene.

Next, we collect the cells by centrifuging after they have had time to produce our desired spider silk.  We lyse the cells and then extract the proteins.  Many undesired proteins (those that are naturally produced by E. Coli) can be eliminated by heat treatment followed by centrifugation—fortunately for us, spider silk (unlike most proteins) is heat resistant, allowing us to discard the pellet and keep the protein extract.

However, our spider silk at this point is still impure.  So, we take advantage of another spider silk attribute: its affinity to metals.  Using HPLC and a column of immobilized nickel ions, we isolate our protein on the column and then strip it off using imidazole. We use dialysis to remove the imidazole wash from the protein.  Finally, we freeze dry the spider silk, and at this point, it is ready to literally be extruded, or “spun,” into fine threads.

According to Western blot analysis, these threads are similar to and have many of the desirable attributes of the silk produced by spiders.  However, even after all this work, it is impossible to perfectly replicate this natural wonder.  Perhaps our results will improve as different plasmids are used and parts of the protocol are modified, but for now, this is one of the many secrets of nature that we have yet to truly unlock.

 

July 2, 2013

The masterpiece of Brazilian culture and history is intricately woven from a captivatingly colorful, sometimes paradoxically contradictive, and always-unpredictable array of variegated threads.  Although Brazil is one of the most ethnically diverse nations in the world, it was also the last country in the New World to abolish slavery.  While Brazil recently enjoyed prosperity under the wildly popular President Lula da Silva, Brazil’s past has been marred by dictators so corrupt that one of them even claimed his own life as his scandalous behavior began to surface (plus, even Lula’s and the current presidencies have not been immune to corruption).  Brazil is the 7th wealthiest nation in the world, but the poverty-stricken war zones of the favelas that permeate large cities like Rio de Janeiro would suggest otherwise.  Brazilians are often stereotyped as frenetic soccer fans that would like nothing more than to have the World Cup in their backyard, though the wildfires of demonstrations that have been blazing through the country this month indicate that Brazilians have other priorities.

But what do all these contrasts in politics and history have to do with energy?  Last weekend, Harini and I visited the Itaipu Binacional, the largest generator of renewable energyin the world. The numbers are impressive—but seeing, hearing, and feeling the thundering mass of water cascade through the hydroelectric dam was absolutely awe-inspiring.  As one of the seven wonders of the modern world, the dam is surely an engineering feat worthy of respect.  However, it was the politics and economics behind the behemoth that really made it possible for about 90% of Paraguay’s and 20% of Brazil’s energy to come from this renewable energy source.  Since the dam takes advantage of a natural resource that lies on the shared Paraguayan and Brazilian border, an enormous effort went and still goes into ensuring equality of the costs and benefits associated with Itaipu.

The dam displaces the need for over 400,000 barrels of oil per day—but at what cost?  For starters, almost US$25 billion were required to construct Itaipu, an investment that left both countries in deep debt.  But the dam isn’t necessarily an environmental boon, either.  Over 700 square kilometers of forest were lost, resulting in the extinction of many plant species and the relocation of Guarani and Tupi natives.

The Brazilian people are very aware of trade-offs and opportunity costs like these.  The aforementioned World Cup inspired the renovation of the National Stadium here in Brasília—a futuristic stadium that now harnesses the sun, wind, and rain to produce up to 2.5 megawatts of energy—a project that cost almost a billion dollars. Almost all Brazilians agree that this money would have been better spent on education, health care, and infrastructure improvements.  For this reason and several others, Brazil has “woken up,” igniting the largest demonstrations the country has seen since its dictatorship years.

Fortunately, my day-to-day work is ethically straightforward.  However, what it lacks in moral intricacies is more than made up for by its scientific complexity.  While still dabbling in several of the other ongoing projects here, I am focusing mostly on oil-content optimization with Alex.  We have been analyzing hundreds of soybeans using gas chromatography-mass spectrometry (GC-MS) to determine which ones have oil profiles indicative of successful gene placement.   Before we can use the GC-MS apparatus, we prepare each sample using a derivatization reaction.  This reaction is necessary because it increases the volatility of the analyte (as the name implies, gas chromatography requires that the components of the analyte be capable of entering a vapor phase to pass through a column).  The samples are reacted first with potassium hydroxide in methanol, and then sulfuric acid in methanol.  Then, hexane is added, and the organic phase (containing the fatty acids we hope to quantify) is extracted and passed through GC-MS.

The oil from soybeans is primarily composed of palmitic, stearic, oleic, linoleic, and linolenic acids.  Greater oxidative stability and decreased viscosity, desirable attributes for biodiesel applications, can be achieved by maximizing oleic acid content while minimizing the presence of the other acids.  GC is employed to separate each component based on volatility differences.  The more volatile acids will spend more time in the mobile vapor phase and elute more quickly than the less volatile acids, which only really enter the mobile phase as the temperature is ramped up.  The apparatus quantitatively detects the presence of anything but the solvent.  Integration over the spectrum of peaks that results enables us to determine how much of each acid is present, thereby allowing us to keep the good soybeans for use in future sexual crosses, and throw out the ones that failed to express the genes we want.

As soybeans and other alternative fuels continue to improve, they will continue to displace the burning of fossil fuels.  However, like the eco-friendly national stadium that came at the cost of public health and education, and the Itaipu Dam which destroyed forests, homes, and national coffers, even the work we do at Embrapa has its share of ethical dilemmas when the big picture is considered.  While displacing fossil fuels will decrease carbon emissions, the increasing amount of land dedicated to soy crops is resulting in the destruction of large sections of the Amazon rainforest, the world’s jewel of biodiversity.  There is no silver bullet solution to the world’s energy problems.  We can only hope that a carefully selected blend of many different green threads will find their way into the fabric of the world’s cultural masterpiece.  After all, silver would be too expensive.

 

June 11, 2013

Ten days have passed since I arrived here in Brasília, Brazil.  Like any other research environment, there are no lectures, no tests, and no homework assignments.  Regardless of the lack of these traditional learning mechanisms, I feel I have learned more about genetic engineering, the Portuguese language, and the Brazilian culture and political environment in these ten days than I could have learned from an entire semester of classes at even a rigorous and prestigious university like Cal.

Harini and I are involved with several of the ambitious projects that are being investigated here at Embrapa’s Genetic and Biotechnology research lab.  Soy is  extremely versatile, with a wide range of industrial, commercial, and medical applications.  Some of the projects we are working on include genetically engineering soy to produce spider silk (a material with tensile strength comparable to that of Kevlar), a chemical that could be used to prevent transmission of HIV, and biodiesel with an optimized oil content profile.  Natural soy is great as a food source—but with subtle changes of its genetics, we can trick soy into doing and being much more.

At first, some of the differences between the lab environment here and the one I am used to (at LBNL) were a little frustrating.  For example, the pace of the work here is often slowed by unavailability of the best supplies and equipment, and breaks from work (for chatting, coffee, etc.) that initially seemed a little too frequent.  However, while there are many reasons why an American lab might seem superior, I quickly realized that for each of Embrapa’s shortcomings, there was a corresponding strength that could and should be replicated in the U.S.  The recurrent breaks in our lab (which I now realize are often necessitated by time-intensive processes like bacterial incubation, induction, gel electrophoresis, etc.), along with the always-amicable ambience that is so prevalent in the culture here, create a work environment so rich in camaraderie that it is remarkably synergetic.  When one researcher’s experiment goes wrong here, he knows he can rely on the collective knowledge of several others to figure out what to fix before running another trial.  Everyone is very aware of what their proximate colleagues are investigating, because the collaborative spirit here is much more powerful than anything I have experienced in the teams I worked with back home.  Meanwhile, the all-too-common Brazilian saying “sempre tem um jeito” (a phrase that describes a way of life as much as it defines a reaction to a particular situation, and is literally translated as, “There is always a way”) provokes a cleverness that not only enables researchers to overcome the challenges posed by inferior equipment and sporadic supplies shipments, but also fosters the kind of insight that is so necessary for experimental design.

Neither the American nor the Brazilian lab environment is “better” than the other.  But perhaps the same overarching principles of genetic engineering we are applying towards soybean genetic modification should be applied to build more efficient and powerful research teams.  How can I select the most desirable traits of my Brazilian work environment and insert them into the genome of my team back in Berkeley?  A cliché comparison, yes, but this is one of the most important questions I hope to answer during my stay here.

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