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Summer 2014 Blog - Joshua Moss

Joshua Moss is spending the summer at Embrapa in Brazil

July 30th, 2014

As I sit here in the Brasilia airport, waiting to board my flight back to San Francisco, I can’t believe how quickly my time in Brasil has flown by. While I’m excited to get back to Berkeley to start my last year of college, I can’t help but feel that I’m leaving just as I got into the rhythm of life in Brasilia.

I was able to be a bit more independent in the lab these past two weeks, so I performed a mini-prep by myself to replicate some of the artificial chromosome DNA we have in the lab. This process involves transforming a harmless strain of E. coli with the synthetic DNA, culturing the cells on an antibiotic-tainted petri dish, selecting bacterial colonies that have survived on the plate due to the antibiotic resistance encoded in the synthetic chromosome, growing the bacteria in a special growth medium so they can replicate the DNA, harvesting the E. coli, and then finally extracting the DNA from the cells. As you probably know, cells contain a lot more than just DNA, so extraction process includes many steps to separate the DNA from RNA, proteins, and carbohydrates that are also in the cells. In the end I was able to obtain a good amount of DNA for the lab to use after I leave.

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The white material at the very tip of the tube is pure synthetic DNA!

Other than that, a lot of lab work has been spent analyzing the CRISPR-CAS DNA so that the lab can ship it to the US so that it can be used to transform Arabidopsis plants. Arabidopsis is a model organism which means that it allows researchers to study the effects of their genetic changes quickly and easily. However, before the DNA could be sent for the transformations, we needed to make sure that the DNA sequence was correct. To do this, my two research heads, Gabriel and Cintia, and I had to come up with three different checks using restriction enzyme digests. The restriction enzymes cut the DNA at specific sites, so once we run a gel after the digestion is complete we can distinguish the lengths of the DNA fragments made through the cuts. If the cuts are the lengths we predict, we can be fairly certain that the sequence is correct.

The main highlight of the past two weeks was definitely the trip I took to Rio de Janeiro two weekends ago. Rio is an incredible city with a lot of history, but what I loved most about it was its natural beauty. The first day I was there I hiked up Corcovado, a half-mile tall mountain right in the middle of the city that has Cristo Redentor (the statue of Jesus overlooking the city) at its peak. That hike has to be one of the steepest hikes I’ve ever done; in some places I needed to climb up steep rocks using only small chains embedded on the rocks’s surfaces. However, once I reached the top the view looking down almost a kilometer above the city made it all worth it.

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Pão de Açúcar and the Botafogo neighborhood on the left, and a partial view of the Atlantic and the Tijuca Forest on the right.

The best was still to come though as I went to Pão de Açúcar (aka Sugarloaf mountain) that night to watch the sun set over Rio. After taking a gondola ride to the top of the mountain I got to watch one of the most beautiful sunsets I’ve ever seen. This view alone makes me want to jump on a plane right now back to Rio.

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This is easily one of the most amazing views I’ve ever had the pleasure to enjoy.

The next day I was able to see some of the famous cultural sites that Rio has to offer like the Arcos de Lapa (a pristine white aqueduct over 250 years old) and the Escadaria de Selaron (a huge outdoor staircase completely covered in beautiful mosaics), but my favorite part of the day was walking along two of the most famous beaches in Brasil, Praia Copacabana and Praia Ipanema. I seriously can’t believe that people live right across the street from this amazing view.

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Walking along the famous Ipanema Beach on my last morning in Rio. Somehow, this is what winter in Rio looks like.

Over the past two months I’ve participated in cutting-edge genetic engineering, learned new lab techniques, become an expert in gel electrophoresis (soooo many gels), made countless new friends, hiked through waterfalls, visited Rio de Janeiro, climbed a mountain through an endangered rainforest, eaten chicken hearts, learned Portuguese (albeit not very well), went to a World Cup match, and celebrated my 21st birthday. I can definitively say that my experiences in Brasil have been invaluable to my scientific and cultural education and that the memories and friends I’ve made this summer will stay with me my entire life. Tchau Brasil, te vejo em breve!


 

July 14, 2014

Another two weeks have flown by in Brasil, and although they passed quickly, a lot has happened. Two weeks ago some soybean embryos were injected with an artificial chromosome in an attempt to see if the chromosome can be incorporated into the soybean’s genome. The embryos are growing quickly and are almost ready to be planted in a greenhouse for further growth. After that they will be analyzed to see if the chromosome was in fact incorporated into the plant’s genome or if it is simply acting as a separate plasmid. It will take months for this analysis to be completed and for the plants that were successful in incorporating the chromosome into their genomes to be cross-bred, so unfortunately I will not be able to find out whether the project was a success (that is until I read about it in a scientific journal).

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Although this plant isn’t a soybean plant or transgenic, this clone demonstrates the end result of a cross-breeding project.

I’ve also been able to help in other areas of the lab such as by helping with an experiment on engineering soybeans to produce higher quantities of oils used to create biofuels. This genetic engineering is not accomplished through the CRISPR-CAS system I have previous talked about but rather through use of TALENS (transcription activator-like effector nucleases) which are engineered to bind to a particular DNA sequence and cleave the DNA allowing for indel (insertion/deletion) mutations to arise. My help on this project had previously consisted of helping transesterify (add ester groups to the end of long-chain fatty acids) the oils produced by the transgenic soybeans, extracting them from the soybean seeds, and running them through a GC-MS apparatus. This machine (which most of my atmospheric chemistry research in Berkeley uses) allows us to determine the identities and rough concentrations of any and all biofuels produced. My help these past two weeks consisted of helping transform bacteria with the altered soybean DNA, plating the transgenic bacteria on petri dishes, and extracting the DNA from the colonies grown on the petri dishes for sequencing.

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Plating bacteria transformed with TALEN transformed DNA

In non-lab news, the World Cup ended this past weekend which was a bit bittersweet because although it was very exciting, the World Cup had become such a huge part of life for me here I was sad to see it end. At least many Brazilians were happy to see Argentina lose to Germany even after Brasil’s resounding 7-1 loss to Germany in the semifinals. At least now that the World Cup is over it will be much easier for me to travel a bit because hotels and planes aren’t as crowded, and I’m planning a trip to Rio de Janeiro next weekend! I was also able to continue exploring the area around Brasilia and went on a few hikes the past couple of weekends. Trust me, I love the Bay Area’s foggy weather, but being in Brasil definitely has its environmental perks.

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Tired after 10 km hiking, but still enjoying a semi-tropical forest at Indaia


June 30, 2014

It’s hard to believe that my internship here in Brasilia is already halfway complete, but looking back I realize that the past month has been packed with research and exploration. The past two weeks in the lab have been focused on replicating and increasing the amount of synthetic DNA we have available to perform bioballistics in the coming weeks. Two weeks ago we received great news that the synthetic chromosome that my research leads had designed had been successfully integrated into a bacterial colony’s genome. Gabriel, my main supervisor here, was really worried that the transformation of the bacteria hadn’t worked because only one colony grew on the petri dish from the transformed bacteria, but thankfully everything turned out fine. We were able to confirm that the DNA was identical to what it was supposed to be through DNA sequencing analysis (the lab itself doesn’t have sequencing capabilities so we had to ship the DNA all the way to South Korea!).

After we got confirmation that the DNA was correct, we began performing “maxi-preps” which means that we transformed large amounts of bacteria with the synthetic DNA and let them replicate in order to produce more of the synthetic chromosome. We then went through the arduous process of lysing the cells, extracting the DNA, and purifying the DNA so that we have as much DNA as possible for bioballistics. It takes one microgram of DNA to perform bioballistics on a soybean embryo, and while that doesn’t sound like much, we are only able to obtain microgram quantities of DNA from any given maxi-prep.

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A lot of the DNA we’ve synthesized over the past month related to the CRISPR-CAS editing project.

After a month of working at Embrapa I’ve begun to notice some striking, but not necessarily unpleasant, differences between how research is conducted here and in the US. For example, in the US we don’t have to wait very long for reagents or equipment to get shipped to the lab. However, here in Brazil researchers sometimes have to wait for weeks or even months for packages to arrive. We are still waiting for something called a Gibson Assembly to arrive which will help us piece together many small fragments of DNA to create a recombinant plasmid, and even though we ordered it a month ago it has yet to arrive. Another difference between here and the US is that many projects seem to overlap with one another. For example, one of the projects I’m working on is using CRISPR-CAS systems to edit soybean genomes, but there are other researchers working on a project to use CRISPR-CAS to edit Arabidopsis genomes while yet another researcher uses a different genome editing method called TALON to edit soybean genomes. The overlap allows many of the researchers to closely collaborate with each other and directly help each other’s experiments.

In other news outside the lab, Brazil continues to amaze me. Two weekends ago I went hiking in a semi-tropical forest where I saw about 30 incredible waterfalls. The hike itself was very strenuous, but the scenery was just what you’d picture a Brazilian river forest to look like. Additionally, I was fortunate enough to attend a World Cup match in Brasilia where I saw Portugal defeat Ghana and secure the US’s place in the knockout rounds! The World Cup really has become an all-consuming aspect of life here for me because not only do people constantly talk about the games, but most stores and restaurants close whenever either Brazil plays or there’s a match in Brasilia. You’ll ever hear me complaining about that though because having the opportunity to work for two months in a country that is soccer-crazy adds enormously to this already enriching experience.

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Watching Christiano Ronaldo score for Portugal at the Estadio Nacional!


June 15th, 2014

My first two weeks in Brazil have been a whirlwind of research, learning, the World Cup, and being a really obvious tourist. Lab work has been going very well even though we are waiting for a lot of reagents to be shipped to us from the US so that we can perform more complex reactions. I have been working mainly with two graduate student researchers named Gabriel and Debora who have been both helpful in teaching me about their research and in taking me around Brasilia. The work we are doing is centered on genome editing using CRISPR-CAS systems partially pioneered by Professor Doudna at UC Berkeley. You can imagine my surprise when Cal managed to find me even when I am 6,000 miles away from campus! Although a lot of research has been done on the CRISPR-CAS system, we are attempting to implement it in soy beans so that they can produce a wide variety of products through relatively simple gene additions. Once we have verified that the vector containing the DNA coding sequences for the CRISPR-CAS system is correct we will begin creating the transgenic soybean embryos. This process may be one of the coolest scientific techniques I have ever heard of. In order to get the vector into the embryos the DNA is actually shot into the seed at a speed of up to 1,500 km/h in a process known as bioballistics. I’ve heard that the procedure itself is not that exciting to watch, but I can’t wait to see how it goes.

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A basic diagram of how the CRISPR-CAS system works. The Cas9 endonuclease (with the help of guide RNA) can identify a target sequence of DNA and cleanly cut both strands of DNA at the target site.

Some days we don’t have a lot of work to do because we are waiting for chemicals to arrive, so I’ve been able to talk to other researchers in the lab to learn more about their work. I actually helped one researcher prepare almost 100 samples of biofuel created from soybean oil. She is trying to use a genome editing technique to prevent the conversion of short-chain alkanes into longer alkanes in order to have higher biofuel yields after transesterification of the alkanes. I actually performed a similar experiment during my freshman year at Cal so I was very interested to go more in depth into the process of biofuel creation.

I’ve really hit the jackpot by coming to Brazil this summer not only because the research is exciting and cutting-edge but also because the World Cup just started here! Unlike most Americans I am a soccer nut and played soccer for more than ten years before coming to Berkeley. A friend I made in the lab took me to a viewing party for the opening match that Brazil played in, and I was shocked by the intensity and fanaticism I encountered. I knew that Brazilians love soccer, but watching the game with them definitely ranks among the most exciting and terrifying moments of my life! I can’t wait to see how the tournament ends, and I hope that Brazil (and France and the US!) keep winning. Outside of the World Cup I’ve also been a super-tourist visiting all of the major government buildings in Brasilia with my new friend Thaina. Brasilia was a planned city and was completed just a little over 50 years ago. As such, all of the buildings which were designed by famed architect Oscar Niemeyer are very modern and beautiful. I can’t wait to continue my excursions around Brasilia and Brazil!

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Me in front of the National Congress

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