On this symposium, which took place in the Biotechnology Development and Training Complex on March 21st, all the partipants were able to share their findings during their 3 semesters of lab work.  It was impressive to see the size of the program and how excited the participants were about finally showing the product of their dedication to science.

In this activity, we served as “judges” of other poster presentations by some of our peers.  This allowed us to get a sense of the scope of  areas of research and topics.  For example, one poster that I evaluated described the used of nanotubes to treat post-stroke injuries.  This poster was presented by Roy Suárez.  This project was interesting in the sense that introduces a novel way of treating patients with stroke injuries after the incident and decreasing the effects of miscommunication between affected areas of the brain.  This is an alternative to stroke therapies that only work if patients are treated in a short time-span after the episode.

On a totally different area, Arnaldo Carreira was involved in characterizing the activity of the inositol 1,4,5-triphosphate receptor.  This receptor is relevant for the decrease in immunological tolerance on human cells that may trigger autoimmune deseases.  This is an important project due to the high incidence of autoimmune deseases in Puerto Rico like diabetes mellitus 2.  Due to my experience in a Ca2+ signalling lab, I was curious about the effect of Ca2+ concentration on the activation of the Ca2+-calmodulin dependent calcineurin.  This phosphatase is crucial in cell response to Ca2+ concentrations that affect cell differentiation.  Specifically for this project, it good to understand how the inositol 1,4,5-triphosphate is also involved in the process.

The last poster I was able to evaluate, was from Samuel I. Ares from Dr. Dimuth Siritunga’s lab.  As a plant biotechnologist, Dr. Siritunga is interested in the control of cyanide concentration in cassava, a starchy tuberous root commonly present in the diet of poor farmers in some developing countries in Africa.  A diet high in cassava that is not cooked in the right way, may contribute to a high absorption of cyanide by the body.  This could be fatal to those that rely on this viand for their nutrition.

In February of 2009, the project moved ahead since all the materials and methods where already available to perform the preliminary experimental procedures.  However, due to my young experience working with filamentous fungi, performing the experiment for the first time did not go exactly as expected.  Inoculation of liquid cultures of P. chrysosporium, requires acquisition of fresh spores as a seed for pelletization.  This process involves filtering and quantification of spores that required new aseptic techniques which I had not experience before.  My first trial ended up with some bacterial contamination when culturing cells, however, I observed growth in both rich and basal media, which is one of the main goals of my project.  This proved that the experiments performed by Jimenez-Tobón et al. could be replicated, which is essential to achieve the main objectives of the project.  Still, there is work ahead that needs to be done in order to reach the final objectives.   I would say that a 3 would be a good number to summarize the status of my project.

A new year has come!  And at the same time there is plenty of work that has to be done, now that all the materials neccesary are available.  Our goals for this semester are:

• Successfully grow Phanerochaete chrysosporium in both rich and basal medium to observe non-granulated vs. pelletized cultures.
• Perform a protein expression screen to observe differential expression related to pelletization.
• Identify proteins that are being over-expressed or inhibited by the basal medium.
• Perform enzyme kinetics assays (if neccesary) to characterize unknown proteins.
• Explore which variables must be changed in order to reduce the size of the pellets to the optimum diameter in which the mass transfer and enzyme kinetics become efficient for possible industrial applications of filamentous fungi.

Last semester we were able to design the experimental setup, perform literature search, and ordering materials and equipment.  The orders where received during the last weeks of the semester, thus, we are now well poised to perform the experimental setup.  The initial steps of fungal growth and media preparation have already started and some weeks are needed to have a culture ready for media inoculation.  After this steps, the observational and molecular assays will be perform to comply with the semester goals.

This semester we will be testing how the white rot fungi grows in the liquid media described by Jimenez-Tobón et al. (Enzyme and Microbial Technology, 1997).  This involves digital imagery and measurement of pellet size.  Also, the protein assays will be performed using SDS-PAGE to observe, by protein staining, the protein expression patterns of both rich and basal cultures.  Identification of proteins of interest will be performed by LC-MS-MS in collaboration with the University of Wisconsin at Madison.  Future tests will be performed in accordance with the initial results.

The current search for environmentally friendly methods for waste disposal, detoxification, and alternative fuel sources, among others, filamentous fungi have become useful tools for the development of novel industrial bioprocesses. Their morphological and cell wall properties confer adequate characteristics suitable for large-scale culture environments that increase productivity. Still, the size of mycelial clumps has to be minimized in order to increase mass transfer efficiency. Characterization of candidate proteins associated to granulation of the white-rot fungi Phanerochaete chrysosporium, will serve as a starting point for the optimization of fermentation cultures of this microorganism. The proposed method of protein analysis is by SDS-PAGE electrophoresis that will show candidate differentially expressed enzymes that will be further analyzed for identification and manipulation. On the following semester, the proteomic analysis will be performed and analysed in order to identify the increase in expression of certain proteins.  Once this work is done, further analyses include protein characterization, pellet size optimization, and kinetic assays, if neccesary.  This characterization will provide important information necessary for a highly efficient and cost effective scaled-up bioprocess for P. chrysosporium harvesting.

White-rot fungi Phanerochaete chrysosporium

As of today, the preliminary procedures of the project have being started, that include Phanerochaete chrysosporium culture preparation.  Materials and equipment necessary to start the experimental procedures has been ordered and I am waiting for their arrival.  For this reason, the level of progress of my project is still a 2 since the essential equipment has yet to arrive.  When I get to start the experimental procedures, we expect to identify those proteins that show an increase in expression after pellet size reduction.  This would increase the efficiency and cost-effectiveness of industrial bioprocesses that use fillamentous fungi.  Harvesting the white-rot fungi in a large-scale process is neccesary for use as toxic chemical  and lignin degradation for fuel production and waste disposal.

Phanerochaete chrysosporium Culture Preparation

A technique tha will be used in my project is the Sodium Dodecyl Sulfate- Poly-Acrilamide Gel Electrophoresis (SDS-PAGE).  This technique consists in the separation of a protein sample that migrates through a porous matrix that controls the migration of its components according to their sizes when exposed through an electric field.  The protein sample is treated previously with SDS that unfolds proteins by hrydrophobic interactions and gives them a net negative charge proportional to its size.  This allows the multiple components of a protein extract to migrate at different rates according to their sizes that may be visualized by Coomasie Blue staining.  This technique will help us to identify differential expression on two samples treated at different conditions.

As of today, my project has being in the process of design, preparation of lab equipment, ordering, and the wait for the materials neccesary for the experiment.  In a scale of 1 through 5 the progress would be at 2. Since I’m part of a young microbiology Lab in a chemical engineering department, to have the neccesary materials and equipment to perform biological samples has been an arduos process.  However, this has provided ample time for establishing a method based on published literature in order to reach our project goals.

     In the Microbial and Biochemical Technology Lab in the Chemical Engineering Department at the University of Puerto Rico at Mayaguez we are interested in finding organisms capable of degrading plant biomass from lignocellulosic plants in order to produce useful compounds like ethanol or lignin-derived products.  My project consists on protein characterization of media that reduces pelletization of the fungi Phanerochaete chrysosporium.  We are interested in finding some target proteins that are resposible for this globulization for later characterization of their enzyme kinetics.  This may be useful for future industrial applications, development of new energy sources, waste treatment, and other applications.

     This summer I had the opportunity to perform research at Columbia University in New York City as part of the Graduate School of Arts and Sciences – Leadership Alliance Summer Research Program.  I was a member of Dr. Brent R. Stockwell’s Lab that searches for novel cell death pathways.  My work involved the characterization of protein expression patterns from cells that were treated with small compounds identified through high throughput screens for the induction of new cell death pathways distinct from apoptosis, necrosis, and autophagy.  A difference in expression was observed for proteins involved in apoptosis like Bid and Caspase-3 among other proteins.  Protein expression patterns differed from those of cells treated with necrosis and autophagy inducing drugs and markers.

     Being in New York City allowed me to enjoy many new experiences aside from bench work.  We were able to attend to different activities like Shakespeare in the Park, The New York Phillarmonic at Central Park, the Museum of Modern Art, Governor’s Island, Broadway shows, and many more…

This semester has brought some advances in the research project but also some setbacks that have slowed down the research.  I have been involved in using a new technique for the creation of truncated proteins for the expression of recombinant proteins.  Some problems with ligation reactions slowed down the process of design of a new construct that would allow the expression of the truncated molecule.  However, lately, the ligation turned out to be more successful when different enzymes were used, longer incubation time were utilized, and new transformation techniques, like electroporation, were used.  I was able to gather more experience in the process of construct design, transformation techniques, ligase manipulation, and troubleshooting negative results.  Also, presentation skills were used and reinforced during this semester.  For next semester we expect to continue with the expression assays now that we may have a functional construct.

Visiting other biopeers’ sites allowed me to get to know their research projects and  know the diversity of projects available at my campus.  I was able to ask questions to other peers about their research and discuss differents aspects of their projects.