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Summer 2014 Blog - Jeanine Porzio

Jeanine Porzio is spending the summer at the Lawrence Berkeley National Lab

July 25th, 2014

Hello again! Next week will be my last week here at the Lab, and I will be sad to leave. In the past two weeks, as always is the case, I have continued to improve and add new features to the V2G-Sim user interface. By now, it is a fairly sophisticated tool, and I am happy with the way it has turned out so far. This week my advisor gave me two big new features to add, so I’ll be trying to get all the kinks worked out next week. My advisor’s going to be in Toronto all next week (3 hrs ahead), and Caroline is back in France (9 hrs ahead), so collaboration is a bit difficult right now. Hopefully I can make it work.

I have made much progress on the website. I designed the whole thing on a hosting platform called Squarespace, but then we found out that we needed to be using Google sites. My advisor and I worked together to make the Google site look as similar as possible to the Squarespace site, and then I spent last week moving all the content over. The structure is completely set up, so all that’s left now is for my advisor to write the content to be displayed on the website.

I’m also trying to finish writing the User Guide and Developer Wiki before I wrap up my work here. The User Guide is nearing completion, but it’s a bit of an uphill battle because as I write the instructions, I am changing the layout of the user interface and improving functionality. So the User Guide constantly needs to be updated, but the basic instructions are there. I’ve been spent a lot of time creating user interfaces (at this internship and in the past) so I’ve been racking my brain trying to come up with all the little quirks and best-practices that are second nature to me now, in order to record them in the Developer Wiki. This way I can try and keep the user interface consistent when the next person works on developing it after I leave.

I’ve also had a bit of fun: as Carielle mentioned in her latest blog , last week we went on a tour of the Molecular Foundry and the National Center for Electron Microscopy (NCEM) together. We were the only two interns who were able to make it for the tour, but we had a great time. Basically, the Molecular Foundry and NCEM is where all the nanoscience happens at LBL. The nano-particles/-materials are made at the Molecular Foundry and then they are examined using the powerful microsopes at NCEM. In the Molecular Foundry we were able to look through windows into the different labs and clean rooms and watch the scientists hard at work. Both our tour guides were very enthusiastic and helpful, and I learned a lot. We were able to go inside the cage of one of NCEM’s two aberration-corrected (scanning) transmission electron microscopes (TEAM), which are the most powerful microscopes they have. Many (if not all) of the microscopes at NCEM are so sensitive that even talking can disrupt the measurements. We were only able to go inside the cage of TEAM I because they weren’t actively taking data at that time. Our tour guide said that if we looked at the log from the microscope, we would see a spike in the data just caused by our presence in the cage (our body heat). It is astounding how powerful these instruments are.

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Two views of the LIBRA (scanning) transmission electron microscope 

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View of TEAM I from inside the cage- it’s very tall


July 17, 2014

These past two weeks have been a little different than the previous weeks of my internship because Marcus, the post-doc I am working with, has been gone. (Congratulations to Marcus and his wife on their healthy baby!) What that means for me is that for the past two weeks, I have gotten to plan and work on experiments on my own. Although this freedom is a little bit intimidating because I do not want to mess up the experiments and have to re-do everything, it is also very exciting to be able to do everything myself. These past two weeks have allowed me to realize just how much I have learned during this internship because all of the techniques I have been using these past two weeks are things I did not know how to do before. Also, luckily for me, Marcus has been quite responsive to emails despite being away from the lab and Angelica, a research assistant in the lab, has been very helpful in answering any questions I have so I have actually been able to get a lot of work done.

For the past two weeks, I have continued my investigation of how nitrogen fixation by bacteria changes under different conditions. If you remember from my earlier blog posts, I used an acetylene reduction assay to measure the activity of the nitrogenase enzyme for three strains of nitrogen fixing bacteria under four different temperatures. Because drought involves an increase in both the temperature and salinity of soil, now, I am using an acetylene reduction assay to measure how nitrogen fixation differs at four different salinities. Unlike the temperature experiment, this time I am using strains of bacteria that have the nifH gene fused with the GFP gene. (nifH is a gene that codes for the enzyme nitrogenase which is responsible for nitrogen fixation. GFP is a fluorescent protein.) This will allow me to directly count cells expressing the nifH gene, which will hopefully be a more accurate way to normalize the nitrogenase activity than using AODC or CFUs to get numbers for the amount of bacteria present.

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Plate of R1Gly nifH::gfp2 and R1C nifH::gfp streaked out to isolate colonies for inoculation. 

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NFb media with four different salinities. I think it’s interesting how the color gets lighter with more salt.

I have also been continuing to use PCR (polymerase chain reaction) to amplify the 16s gene for the DNA that I extracted from the rhizosphere and rhizoplane of tobacco and switchgrass seedlings. In my last blog post, I mentioned I had used the 27F and 518R primers to amplify part of the 16s gene. This week, I used the 27F and 1492R primers to see if I could amplify a larger part of the gene so that we will have better results when we do RFLP (restriction fragment length polymorphism) analysis later on. Luckily, I was able to amplify and purify the DNA for each sample!

I also got to go on a tour of the Molecular Foundry and the National Center for Electron Microscopy (NCEM)with Carielle, one of the other Cal Energy Corps interns at LBNL. The tour was arranged by the LBNL undergraduate internship coordinator, but Carielle and I were the only interns on the tour. The Molecular Foundry and NCEM are really cool because they are User Facilities. What that means is that scientists from all over the world can write proposals to use the special equipment at these facilities and if they are approved, they get to use the facility for free! On the tour, I learned about and got to see some of the equipment used for nanoscience projects at the Molecular Foundry and many big fancy electron microscopes at NCEM. Unfortunately, I forgot to bring my camera and as one of the rare people without a smart phone, I couldn’t take any pictures. Sorry!


July 3, 2014

Even though it has only been four weeks, I have already learned a lot from my internship. One of the most important things I have learned is that real science is not a neat and orderly process, but is filled with unexpected results, mistakes, and surprises. For instance, what at first seemed like a simple enough experiment: growing three isolates of nitrogen-fixing bacteria at four different temperatures and measuring how much ethylene is produced by the reduction of acetylene, has become far more complicated than I anticipated. Surprisingly, one of the isolates, R1Gly can apparently produce ethylene without the addition of acetylene. This is very interesting and puzzling because ethylene affects plant growth. According to the literature, ethylene is supposed to inhibit plant growth, but in previous experiments, the post-doc I am working with has found that adding R1Gly to soil enhances the growth of plants. Additionally, in order to normalize the amount of ethylene produced by each isolate so that nitrogen fixation by nitrogenase can be compared between different temperatures, I need to determine the amount of bacteria in each tube, but it has been a process of trial and error to plate good dilutions to calculate colony forming units (CFUs). When I was finally able to get isolated colonies to grow, the CFUs did not agree for the different dilutions I tried. As an alternative to calculating CFUs, for one isolate, R1C, I learned how to do Acridine Orange Direct Cell (AODC) counts to determine the number of cells in my tubes. The dye Acridine Orange stains nucleic acids. When you use a microscope with UV light, you can see cells that have DNA stained green and RNA stained orange.

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R1C cells stained with Acridine Orange.

In addition to investigating how nitrogen fixation differs with different temperatures, I have also been working on another project studying how the microbial community changes during the development of Tobacco and Switchgrass. The plants have been growing well in the greenhouse and I learned how to extract DNA from the soil of the seedlings using the MOBIO PowerSoil kit. To extract DNA, for each Tobacco and Switchgrass seedling, I took samples from the rhizoplane (soil directly in contact with the roots) and the rhizosphere (soil next to the roots but not in direct contact).

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Tobacco and Switchgrass seedlings used for taking samples of the rhizoplane and rhizosphere.

I then used PCR to amplify part of the 16s gene which encodes part of the bacterial ribosome and I used gel electrophoresis to see if I was able to amplify the gene. Fortunately, I was successful!

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Each PCR sample from the DNA extracted from the Tobacco and Switchgrass seedlings has a 500 base pair band that correlates to the amplified part of the 16s gene.


June 19th, 2014

A lot has happened in the two weeks that I have been working in Dr. Romy Chakraborty’s lab at the Lawrence Berkeley National Lab. After getting my official LBNL affiliate badge, I spent the first few days taking online safety training courses. Safety is very important at LBNL and I was not allowed to start working in the lab until I finished all of the safety training. But now that I have finished all of the online training, I have already started working on experiments!

For one experiment, I inoculated sealed tubes containing a nitrogen-free medium with three strains of nitrogen fixing bacteria that Dr. Marcus Schicklberger, the post-doc I am working with, previously isolated from soil associated with the roots of Switchgrass and Tobacco plants. We added acetylene to the tubes and incubated them at four different temperatures. Then, I learned how to use a GC to measure the presence of acetylene and ethylene in the tubes. The presence of ethylene in the tubes is a proxy to indicate that the bacteria use nitrogenase to fix nitrogen because nitrogenase can reduce acetylene to ethylene.

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Tubes of bacteria in nitrogen-free medium

Another experiment that we started focuses on how the microbiome changes during the development of Switchgrass and Tobacco. The post-doc started growing Switchgrass and Tobacco seeds in petri dishes at JBEI so I got to tour JBEI when we went to collect the seedlings to transfer them into soil at a greenhouse. Just after about one week, the plants grew a lot!

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Tobacco seedlings v.s.Tobacco plants after about a week || Switchgrass seedlings vs. Switchgrass plants after about a week

The PI also had me present an overview of what I will be working on this summer at the lab meeting so here is a brief summary of what I will be doing: I will be studying the microbiomes of Switchgrass and Tobacco, which are biomass sources for biofuel. There are multiple aspects of the microbiomes that I will be studying; in particular, I will be studying nitrogen fixing and phosphate solubilizing bacteria from the rhizosphere, so that hopefully in the future, the interaction between these bacteria and plants can be utilized to provide a sustainable source of nitrogen and phosphate as an alternative to fertilizer. Another component of my internship will be determining how nitrogen fixation differs in a changing environment, with a focus on drought as increased temperature and salinity. This research is really interesting because it will give us an idea of how the ability of the bacteria to fix nitrogen will change as drought becomes increasingly prevalent with climate change. Additionally, the PI told me that this project is particularly exciting because no one has precisely asked the questions that we are before! The final component of my internship will be studying how the composition of the microbial community changes during the development of Switchgrass and Tobacco. It is a lot to work on and I am excited to see how this summer will progress.

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