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Summer 2012 Blog - Hank Fanchiu

Hank Fanchiu is spending twelve weeks with the Brazilian company Embrapa.

 

June 16, 2012

Extracting and purifying DNA from multiple strains of E. coli bacteria

EMBRAPA—short for Empresa Brasileira de Pesquisa Agropecuária, or the Brazilian Enterprise for Agricultural Research—has many centers devoted to different agricultural fields. My internship placement is at CENARGEN, the National Center for Genetic Resources and Biotechnology, located in Brasilia, Federal District. Specifically, I am working in the ­­­­Synthetic Biology Lab, assisting in two separate projects involving soybeans.

The first project is the research of fatty acids in soybean seeds. Although soybean seed is approximately 18% total oil, not all the fatty acids available are good oil for producing biodiesel.  In particular, the oxidative instability of soybean oil—therefore the instability of biodiesel—can compromise engine performance.  To enhance stability, the oleic acid concentration in soybean oil needs to increase, while the palmitic acid concentration needs to decrease. This can be done by restricting certain genes in the soybean to produce a high-oleic-acid and low-palmitic-acid phenotype.

At this stage of research, we are using gas chromatography-mass spectrometry (GC-MS) to analyze the oil content of soybean seeds. So far, we have been analyzing stock fatty acids to create standards for the mass spectrometer. We first had to chemically modify some of the fatty acids to be suitable for the GC-MS.  Through the process of esterification, we added a methyl-ester group to each fatty acid, stabilizing the fatty acids and preventing them from oxidizing. The additional methyl-ester group allowed the fatty acids to be more soluble in hexane, the solvent used for the GC-MS. 

A spider can produce various types of silk, for different purposes,
using different glands.

The second project that I'm involved in is the genetic engineering of soybean seeds to produce spider silk proteins. All spiders can produce a variety of silk, each for a different purpose (see figure). One particular variety of spider silk is used as the main frame of the spider web. This type of spider silk has similar resistance as and more elasticity than Kevlar, the material used to make bulletproof vests. Since it is unfeasible to domesticate spiders to mass-produce silk for industrial applications, the goal is to be able to synthetically produce this type of spider silk using soybean seeds to produce the proteins.

The proteins that compose the main frame silk are called the Major Ampullate Spidroin (MaSp). Their large sizes, and therefore the strength of the silk, are mostly determined by the number of repetitions of a specific genetic sequence in the gene. In Parawaxia spiders, two similar genes have been identified to code for the silk proteins. After synthesizing the sequence that is repeated in one of the genes, we can transform E. coli bacteria with this sequence, replicate, and connect copies to create a long repetition. Then, we can transfer this repetition to soybean seeds to produce the proteins. However, matching the true number of the repeats in the gene, thus the true size of the proteins, has not yet been possible. Nevertheless, we are now attempting to increase the number of repeats to increase the size of MaSp.

Having background in biology and chemistry allows me to understand the ongoing research without excessive explanation. Especially with my previous experience working in a crystallography lab, I have been able to perform most procedures with little help or demonstration. Still, since mass spectrometry is a new topic for me, I have been spending much time following my colleagues to learn the research process revolving around the mass spectrometer. The little bit of language barrier only adds more time. I feel at ease working here at EMBRAPA, but I definitely have much more to learn.

 

July 3, 2012

Due to the time constraint of my internship and the nature of research, both my supervisor, Dr. Elibio Rech, and I agreed that ten weeks is not enough time to begin a project and reach any significant conclusion. Therefore, my objective here at EMBRAPA has shifted to assisting in multiple investigations, led by different people. In addition to analyzing the oil content of soybean seeds and transferring genes encoding for spider silk proteins, I have also become involved in the purification and analysis of cyanovirin in soybean seeds.

Cyanovirin  (CV-N) is a protein, produced by the cyanobacterium Nostoc ellipsosporum, that has been shown to inhibit the human immunodeficiency virus (HIV) as well as some other viruses. If this virucidal protein can be mass-produced at a low cost, it can be used as a topical treatment to prevent infection by HIV. Currently, cyanovirin can be synthetically reproduced by E. coli by transforming the bacteria with the cyanovirin gene, but the cost of using E. coli to produce cyanovirin is far too high. On the other hand, cyanovirin cannot be produced by yeast without undergoing unwanted modification, known as glycosylation, that deactivates the protein. In soybean, cyanovirin is produced with an unknown modification that does not appear to deactivate the protein. Our goal, then, is to purify and then characterize the sequence and/or modifications present in the cyanovirin structure produced in soybean seed.

Using the ultrafiltration system to separate soybean proteins
based on molecular size.

The process of analyzing soybean-produced cyanovirin began by grinding selected seeds into powder and extracting total soluble proteins (TSP) from the seeds. We then passed the liquid extraction through an ultrafiltration process using a filter pump, while sizing down the filter after each filtration process is complete, to separate molecules into different size ranges. Next, using high-performance liquid chromatography (HPLC), we selectively separated a cyanovirin-rich fraction based on cyanovirin’s hydrophobicity on a reversed-phase column. With a semi-purified sample containing enriched cyanovirin, we used  matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-Tof-MS)  to determine the molecular mass of the modified cyanovirin extracted from soybean seeds. To determine the antiviral effectiveness of soybean-produced cyanovirin, however, we need a pure sample. What we’re working on, as of now, is purifying the sample further in order to obtain pure cyanovirin for sending to the lab’s collaborator.

Beyond working in the lab, I have been really enjoying my time in Brasilia. In this past month that I’ve been here, I’ve gone sightseeing, eating plenty of traditional Brazilian food, windsurfing, and even learning to play the pandeiro (a tambourine-like percussion instrument). During a particular weekend, I went to central Brasilia to see all the famous buildings, including the [analog] TV Tower (Torre de TV). From there, I could almost view the entire city. Brasilia is certainly a great place to be, and I am trying to get the most that I can out of it.

Panoramic view of central Brasilia from the observation platform of the TV Tower

 

July 24, 2012

Purifying samples containing the cyanovirin protein, using high-performance liquid chromatography (HPLC), is a process that requires concentration and lots and lots of patience. Last week, after my colleague, André Murad, and I had separated a fraction of protein extract from a batch of genetically engineered soybean seeds, we injected the sample—0.5 milliliters at a time—into the HPLC column to further separate the molecules contained in the sample. As the sample passed through the column, the molecules are adsorbed to the inner surface of the column due to their hydrophobicity. The HPLC system was programmed to increase the concentration of the organic solvent passing through the column, which, depending on the molecule’s hydrophobicity, would cause the molecule’s desorption and flow out of the column. This allowed us to clearly collect fractions of individual types of molecules. Our goal was to collect a pure sample of cyanovirin protein, assuming the protein was even present in the original sample.

But collecting fractions meant manually catching the sample drops flowing out of the column. While the HPLC program could identify when molecules were desorbing by indicating peaks on a graph, the machine could not automatically collect the samples (at least not this system). By superposing a graph of a previous sample run onto the plot of the ongoing flow, we could know when to expect certain desired peaks that indicated when to collect the sample. 

André and I anticipate for samples to flow out of the HPLC column
for collection

For each run of 0.5 milliliters, I waited approximately 25 minutes and 30 seconds—as not every run was exactly identical—for the first desired peak to start forming before holding a tube under the outgoing flow. Drop by drop I collected the fraction, until the peak ended and another began, and then I swapped tubes to collect a new fraction. There were seven desired fractions that flowed out over the span of 15 minutes. After the last fraction was collected, I stopped the run, waited for 15 minutes for the column to be completely washed and re-equilibrated, and injected another 0.5 milliliters. The entire process required roughly 55 minutes, with 10 milliliters of sample total, meaning I had to spend almost 20 hours collecting fractions. 

We didn’t run the sample through the column 20 times. After we had collected enough of each fraction, we analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-Tof-MS) to determine whether the cyanovirin protein was in fact present in any of the fractions. This analysis did not return promising results, so we performed SDS-PAGE for any explanation. The gels were run yesterday, so as of now we are waiting on the results.

From this I’ve learned that, in research, it is definitely possible to not obtain any results, even after spending hours watching samples fall drop by drop. I’ve also learned to remember to go to the restroom whenever I have the free time before I regret not doing so in the middle of a sample run.

 

August 10, 2012

A typical weekend fair in São Paulo’s Japantown,
located in the district of Liberdade

What I didn’t detail in my previous blogs was everything that I was doing outside of the lab. With 10 weeks to spend in Brazil, I planned with the other Cal Energy Corps interns, also my friends—Joanna Ji, Jennifer Jong, and Susan Lee—to travel throughout the country on the weekends. In total, I visited four different cities and regions of Brazil, experiencing some of the best of the country.

Our first trip was to visit Joanna in São Paulo, the largest city in Brazil as well as in South America and in the southern hemisphere. To the locals, São Paulo is the “New York of Brazil,” and indeed it was a bustling metropolis with skyscrapers, districts, and many, many people—completely different from Brasília. In São Paulo is also where the largest Japanese community is located outside of Japan. There, we enjoyed the local Japanese street food and festive atmosphere.

Weeks later, we rendezvoused in Rio de Janeiro, arguably the most beautiful city in the world. Christ the Redeemer statue, Sugarloaf Mountain, Ipanema, Copacabana, the Selaron Steps, the favelas (shantytowns)—we explored them all. What was even more exciting was hang gliding from Pedra Bonita, spiraling over the southern beaches of Rio and being entirely captivated by a certain charm of the city. 

The Itaipu Dam: Renewable energy!

From the towering high-rises of São Paulo to the clean sandy beaches of Rio, Jennifer, Joanna, and I then flew to the city of Foz do Iguaçu to see the majestic Iguaçu Falls. On Day One we visited the Brazilian side of the falls (Iguaçu), as well as the Park of Birds (Parque das Aves) to walk among toucans, macaws, parrots, and other exotic birds. On Day Two, my little knowledge of Spanish helped us navigate to the Iguazú Falls in Argentina. As if we weren’t already soaked from the unexpected rain and the splashes from the cataracts, we paid Argentinean pesos to ride a boat to immediately next to the waterfalls. Upstream from the Iguazu Falls, the water from the Paraná River powers the Itaipu Dam, the largest operating hydroelectric facility in terms of annual energy generation.

To surpass all previous trips, Jennifer, Joanna, and I ventured into the Amazon. Of course, we didn’t trek into the rainforest by ourselves. As part of a tour group, we spent two nights in a lodge and one night in the forest, experiencing some of the most unbelievable things. We ate the grubs of fireflies, fished for piranhas (or rather fed them because we failed to catch any), caught a 7-month-old caiman, spotted a boa, monkeys, and birds, picked a swimming sloth out of the river, and even swam with pink river dolphins. Undoubtedly, the three days I spent in the Amazon were the most extraordinary time of my life.

Our guide found Brazilian nuts (castanha) for us to snack
on during the jungle hike

The lodge was located in one of the villages by Rio Negro, the left tributary of the Amazon. Sadly, for a community of 67 people, there were no funds for any renewable energy infrastructure. To receive electricity, the villagers connected long-distance wires in series from neighboring villages. For water, they filtered river water but used lots of gasoline and diesel for power. They also had to frequently sail to Manaus, the capital city of the state of Amazonas, to buy food, mineral water, and supplies. These trips required over an hour each way, burning liters and liters of petroleum. With the work that the Cal Energy Corps interns are doing throughout the world, I certainly hope that in the near future we can assist villages like these and help preserve the diminishing Amazon Rainforest.

The amazing Amazon is a treasure that we need to help preserve
through renewable and sustainable energy

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