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Summer 2013 Blog - Harini Sadeeshkumar

Harini Sadeeshkumar is spending ten weeks in Brasilia, Brazil working at Embrapa. 

August 20, 2013

During the last two weeks of my internship at Embrapa, Geoff and I have been mainly focused on two things. Firstly, we finished making a membrane containing the digested DNA from soybean plants with modified oil content for southern blot. Secondly, we began work on our posters, addressing any questions we had about our projects and learning the finer details of the design of the transgenic plants we have been working with.

The gene we have mainly been working with takes advantage RNA interferons (RNAi). We want to silence the genes FATB and CN2, involved in regulating the fatty acid composition of soybean seeds, in order to achieve an oil composition that is desirable for biofuel production. We build a gene cassette with a promoter, conserved region in the two genes, and intron region, the inversion of the conserved region, and a terminator. This cassette is integrated into the genome randomly through bombardment. When the mRNA from this cassette is transcribed by RNA polymerase II, it forms a hairpin which is recognized by DICER. This chops up the mRNA into small fragments. The mRNA can then hybridize with the conserved regions in the FATB and CN2 genes. The DICER recognizes double stranded RNA and will cut up those genes, hence silencing them.

When creating a transgenic plant, the ultimate goal is to create a plant that is homozygous for the gene of interest. When we first started working on the project aiming to modify the oil content of soybean, our mentor, Nicolau, explained that the first generation of transgenic plants would be obtained when the apical meristem of non-transgenic soybean plants would be bombarded with a plasmid containing the gene of interest using a biolistic particle delivery system, which is basically a gene gun. Once the bombarded apical meristems are grown up into soybean plants, they can be screened for the gene of interest using PCR, etc. A single plant can contain multiple copies of the gene, some or all of which can be non-functional or truncated. ELISA can provide a good indication of whether the gene is functional or non-functional. Once plants containing a functional transgene are identified, a homozygous line needs to be created. The lab’s initial strategy had been to allow the plant to auto-pollinate, but this resulted in difficulties because the homozygous and heterozygous lines cannot be differentiated. Therefore a new strategy was developed in which transgenic plants from the F1 generation would be crossed with non-transgenic black soybeans to create a hemizygous F2 generation. Members of the F2 generation that are found positive for functional copies of the transgene are auto-pollinated to create an F3 generation.  Positives obtained in the F3 generation can either be homozygous or heterozygous for a functional copy of the transgene, but when they are auto-pollinated, positives in the F4 generation are homozygous. This entire process is very time consuming, and can anywhere from several months to several years. Throughout the summer, Geoff and I have screened plants in the F2 generation of the plants with modified oil content, but it will at least 2-3 months before the F3 generation will be ready.

rna_interference_mechanism

Mechanism of RNA interference. Baulcombe, David. “RNA Silencing in Plants.” Nature 431 (2004): 359. Web

 

July 23, 2013

Summer is flying by, and Geoff and I are making progress in lab and continuing to explore Brazil.

macerating_tissue_harini

Macerating plant tissue for genomic DNA extraction.

At Embrapa we have begun a southern blot experiment to verify the presence of a transgene coding for CVN (protein that inhibits HIV entry into cells) in two soybean plants. We had previously detected the presence of the CVN transgene in these plants using PCR on genomic DNA extracted from the plants, but southern blotting provides a more conclusive check and also allows us to detect the number of functional copies of the gene in the genome. A southern blot experiment basically involves running DNA on a gel, transferring the DNA from the gel onto a membrane and detecting the presence of a specific fragment through hybridization to a radioactive probe.

To prepare the DNA that is to be transferred to the membrane, we must purify at least 15 µg of genomic DNA from each plant of interest. This is a rather large quantity of DNA and requires us to start with 3 or 4 leaves from the soybean plant. We must macerate the leaves into a very fine powder using liquid nitrogen in order to maximize the amount of genomic DNA we can extract. After extracting the genomic DNA using a kit, we need to linearize it with a restriction enzyme that only cuts once, because Southern Blot only works with linear DNA. Then we must run the gel very slowly overnight to prevent curvature of the bands. Once we have the gel, a few more steps must be completed before the transfer of the DNA to the membrane that causes the DNA to denature. The DNA must be single stranded to allow for hybridization to a probe. When the gel is finally ready, it is placed on top of the membrane in a set up that ensures that it is always soaked with a special transfer buffer. After leaving this set up overnight, the DNA will transfer into the membrane. In order to fix the DNA in the membrane, it is left at 80°C for 2 hours, and the membrane is finally complete. It took us about a week to set up the membrane and to create the probe, however we have not yet had the opportunity to radioactively label the probe and complete the experiment.

southern_blot_harini

Setting up the membrane transfer step of the southern blot.

It has also been an exciting time to be at Embrapa due to some major progress being made on another one of the lab’s projects regarding the production of spider silk proteins in E. coli. For the last several months, the lab members working on this project have been growing up their transgenic E. coli, inducing it to produce spider silk proteins, harvesting the proteins and purifying them. Just recently they performed an experiment for the first time, to spin the harvested protein into fibers and it was a great success.

Outside of Embrapa, Geoff and I have managed to travel to different places around Brazil every weekend. Since my last post, we have visited Rio de Janeiro, Chapada dos Veadeiros National Park, and the Amazon Rainforest, all of which were extremely different environments and wonderful to explore.
July 2, 2013

geoff_harini_turbines

Geoff and Harini in front of one of the turbines in the Itaipu Dam.

It has now been a month since Geoff and I have arrived in Brasilia, and it is safe to say that we are constantly having new experiences and learning a lot.

At Embrapa, we have continued to assay modified soybean plants for the presence of transgenes through genomic DNA extraction and PCR. In addition, we have also learned how to do an ELISA (Enzyme linked immunosorbent assay) to measure the expression level of transgenic proteins of interest. While PCR is useful for determining whether a transgene is present in the genome of soybean in the first place, ELISA is good for determining whether the transgene is located in a place of high enough expression in the genome to be useful. This week we will be learning how to do a Southern Blot which is useful for determining how many functional copies of a transgene have integrated into the genome. For a separate project, we have also been learning how to use GCMS (gas chromatography mass spectrometry) to analyze the oil content of soybean seeds which has been modified to be more suitable for biofuel production. Ideally the seeds should have very high oleic acid content, and low palmitic acid content for improved cold flow and oxidative stability. We have learned both how to prepare samples to be run through the GCMS and to analyze the data.

It has been an interesting time to be in Brazil due to the large social movement that is taking place. Many protests are taking place in cities all over Brazil addressing a variety of issues including the billions being spent on stadiums for the upcoming World Cup and Olympics instead of education, healthcare and public transport, as well as widespread corruption in the government. Geoff and I actually had the opportunity to attend a smaller protest in front of the National Congress in Brasilia. It was peaceful for the most part, and it was interesting to observe the signs people were holding and the chants being said.

iguacu_falls_harini

One view of the Argentinean side of Iguaҫu Falls.

This past weekend, Geoff and I traveled to Foz do Iguaҫu and got a chance to visit the Itaipu Dam as well as both the Brazilian and Argentinean side of Iguaҫu Falls. Touring the largest dam in the world was a fascinating experience. The dam is shared equally between Brazil and Paraguay, in terms of financial contribution, energy produced, and even employment at the dam. Paraguay uses 10% of the energy produced by the Itaipu Dam to meet 75% of its energy needs. Brazil uses 90% of the energy produced by the dam (the 50% it has rights to, as well as 40% purchased back from Paraguay) to meet 16% of its energy needs. The beginning of the tour involved watching a video that was misleading because it only highlighted the positive aspects of the dam including its benefits as a renewable energy source, and even that there is a spawning channel for fish that routes them around the dam so that they aren’t killed. Regardless, pictures do not even show what a wonder this dam is to behold. Iguaҫu Falls was just as incredible to see. Water levels were so high due to heavy rainfall, that some trails around the falls were closed because water was flowing right over them. It was amazing to see both a natural wonder and a modern wonder all in one weekend.
June 11, 2013

harini_greenhouse

In the greenhouse containing the transgenic soybean plants.

It has been a little over a week since Geoff and I arrived in Brasilia. Upon arrival, a student who works in our lab, Gabriel, picked us up and helped us get settled in apartments. Gabriel took us to our lab at Embrapa on Monday and we got a chance to meet everyone in the lab and the PI, Dr. Elibio Rech. He encouraged us to talk to everyone in the lab about their research and learn about all of the different projects going on in the lab.

The goal of the project we will primarily be working on is to modify the oil content of soybean seeds to be more suitable for biofuel production (enhancing oxidative stability, augmenting cold flow, etc.). This is done by bombardment of the apical meristems of soybeans plants with microparticles coated with plasmid DNA, containing genes to modify the fatty acid profile of the plants. If the bombardment is successful, the soybean will integrate the plasmid into the genomic DNA. The lab has already obtained transgenic plants using this technique, but transgenic lines that are homozygous for this transgene must still be obtained. A lot of the work we will be doing is verifying the presence of the transgene in soybean plants using PCR amplification and Southern Blotting. We will also be analyzing the oil content of transgenic seeds using mass spectrometry.

Additional projects we have learned about include modifying soybean to produce a variety of therapeutic proteins such as a coagulation factor and a cancer antigen, and modifying E. coli to produce spider silk protein.

In past lab experiences, I have only had the opportunity to work with transgenic E. coli. It is an interesting change to work with a higher order organism such as soybean, and learn novel methods of genetic modification developed at Embrapa. In addition, I will learn a lot of new laboratory techniques such as Southern Blotting and mass spectrometry which I have never used before. Overall, I expect this experience to expose me to a lot of interesting avenues of research and allow me to develop lab skills to supplement those I have already developed at Berkeley.

After spending a week here, there are two key differences I have observed between my lab experience in Brazil and in the United States. Firstly, I have observed that Embrapa has a generally friendlier and more collaborative atmosphere than labs I have worked in at Berkeley. Everyone in our lab in Embrapa has been greatly approachable since we have gotten here, from our PI and researchers to other undergraduates.  In contrast, while labs I have worked in at Berkeley have also had a friendly atmosphere, it took a lot longer to feel comfortable approaching researchers and grad students. I think this can be attributed to cultural differences between the U.S. and Brazil; Brazil presenting a friendlier atmosphere overall. This welcoming atmosphere at Embrapa has been greatly conducive to learning.

Secondly, I have observed that a lot of things I have taken for granted in labs at Berkeley are unavailable here at Embrapa. For example, because of extremely high import taxes in Brazil, lab supplies such as miniprep kits become unaffordable. While I have always in the past used a miniprep kit to isolate plasmid DNA, here all of the solutions used for minipreps are made by hand. Another disparity is the length of time it takes to order oligonucleotides. While in the United States oligos are delivered within 24 hours of an order being placed, here in Brazil it can take several months for oligos to be synthesized and delivered. I imagine that a lot of forethought is required in one’s experimental planning to prevent having to wait months to continue an experiment. Due to the differences in availability and price of certain lab supplies, I imagine that research is a little slower in Brazil compared to the United States.

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