So this story comes to an end… Or has it? One and a half years ago, 100 students ventured into the world of scientific research sponsored by BioMINDS. Some students had already developed a degree of lab skills, while others were just beginning to acquire them. I belonged to the latter of these two groups. 

Yet after all this time, I have seen how we have surpassed many obstacles, endured numerous hardships, and most of all: acquired valuable knowledge that classes alone cannot provide. We have learned to make better use of our time, we have seen how significant some seemingly irrelevant findings can be, and we have begun to develop a set of analytical skills to interpret real-life data that may someday lead to scientific breakthroughs!

In order to asses our analytical skills and our experience in the BioMINDS program, we attended at the BioMINDS Annual Poster Day, just about a month ago. During this day, we heard and participated in a series of conferences given by leaders in the pharmaceutical-biotechnological  industry, such as Mr. Emilio Rivera, Vice President of Operations at Amgen and Mr. Pablo Vila, Training Manager at Abbott ABL. Also, we shared our experience in this program and got hear what some mentors thought about the program in general and how this initiative has lead to positive outcomes at all the different campuses from the University of Puerto Rico. 

Near the end of the program, all of us, students from BioMINDS, got to present our posters, as well as evaluate three other randomly selected ones. The students I got to evaluate were: Josué Millán, from the laboratory that I worked at last year, Rey Yamil Pagán, also from my college, and Charles Fermaintt from UPR at Humacao. All three made excellent posters. Their presentations were clear and well informed. Here is learned:

Josué’s project focuses on the isolation and characterization of bioprospects from the Pitcher plant Nepenthes sp. Indeed, he had already isolated and characterized a variety of organisms that were capable of surviving highly acidic environments, that are natural to the gooey substance inside Pitcher plants. He also characterized the bioprospects biochemically and was waiting for the results of the sequencing laboratory.

Rey worked on the biological analysis of a biomedical material – Titanium Alloy. He investigated how different fusions of the metal along with other inorganic substances affected osteoblast adhesion to the material. He determined that two of the Ti-fusions were not biocompatible and therefore, not suited for osteoblast growth.

Charles, on the other hand, worked on a project involving gene expression. He intended to identify certain genes expressed by Saccharomyces cerevisiae via the use of repressor genes. His work plays a very significant role in processes such as embryonic development and tumor progression.

At least, that’s where nanotechnology is taking us. Everything from nanowires that absorb oils (right image), to nanoscaled biosensors are examples of innovative materials this young branch in science has produced.  In the past two decades, federal funding agencies, as well as private ones have funded billions of dollars in nano-oriented research. Apart from all the beneficial physical properties nanoscaled systems offer, they have had great progress in many biomedical problems humanity faces today. Liposomes (image below) and biodegradable nanoparticles are examples of nanoscaled systems that are already approved for treating certain cancer types and even menopausal sypmptoms!

            The change nanothecnology is making around the world is analogous to the transition of research laboratory and project topics that I am going through right now. Last semester, I did not mention that the story regarding the search for novel antimicrobials in Puerto Rican soil would end, but it did. Now, I am working at the Biomaterials and Biomedical Engineering Research Laboratory from UPRM’s Chemical Engineering Department, under the mentorship of Madeline Torres-Lugo, PhD and  Héctor Rodríguez, PhD candidate.  Although this sudden change from Biology to Biomedical Engineering (BME) research is radical and unexpected, I believe that it was necessary. The research project I worked on last summer gave me more than just a taste of BME research. It kindled my passion for research and replenished my strengths to pursue a graduate degree. I thank Dr. Carlos Rios-Velázquez and his extraordinary research team for enriching my knowledge in molecular biology and bacteriology and teaching me the skills and strategies to be successful in more than just the lab bench.

This semester I aim at: learning and developing protocols regarding specific in vitro experiments, improving tissue culture skills, fluorescence and light microscopy data analysis, among others. I will find myself replicating a fellow undergraduate’s experiments at random intervals in order to corroborate my skills and have duplicate sets of data.  In parallel, I will be learning about progress on hyperthermic treatment of cancer cells, given that it will be the focus of the research project I will work on. This semester’s work plan has no relationship with past ones, since they are completely different projects.  However, as the title suggests, these may seem like small steps, but they will contribute equally to the project like the pixels in a bigger, better picture.

For all the information regarding my new research project and progress from this entry henceforth, you may visit my other research blog: http://magcan.wordpress.com/ .

I think my previous post merits more explanation. In this research project, as you may have read from my very first post, we intend to complete two objectives: genetic screening for uncultivable antimicrobial production and whole-cell screening for cultivable antimicrobial production from soil samples. Both start by randomly taking soils samples out of areas such as a rainforest, salt lands, and parking lots (incredible!) in Puerto Rico. 

Genetic screening is rather simple in comparison to troublesome incubators and take-too-long-to-grow microbes. In this phase, soil samples are subjected to commercially available soil DNA isolation kits. This DNA extract is then put into a thermocycler  where it undergoes polymerase chain reaction using selective primers that amplify genes encoding for enzymes strongly associated to antibiotic production called PKS and NRPS. In the previous post, I explained that even tough amplification occurred, the primers for PKS were not being used at conditions that optimized their specificity. Hence, we obtained positive results, all samples had PKS amplifications, but further optimization will give us a better idea of the concentration of amplicons and variability within them. 

In another level, we want to search for novel antimicrobial activity from cultivable soil microorganisms. After obtaining soil samples, we suspend them in water, dilute them, plate them on solid media, and incubate them under different conditions. Once the microbial populations start to flourish, we isolate those microorganisms that present antimicrobial activity – ergo, inhibition zones – and test them against our target bacteria. From the results that I posted the other day you can observe that we focused this aspect of our experiment on two actinomycetes isolated by the grad student Vanessa Cardona. The first one, AR, did not present any antagonistic activity over Staphylococcus aureus or Pseudomonas aeruginosa. However, in a very intense week at the lab, every single culture plate belonging to my experiment with AR got contaminated. Guess who? Well Fungi of course! Not only does every microbiology laboratory has to fight against fungal contamination, but  when you live in a sunny, beautiful and immensely humid tropical island such as Puerto Rico, you are forced to take extreme caution with the purity and sterility of the incubators, tools, etc. Well, during Mr. Fungi’s visit to my AR cultures, we were surprised to see that AR was able to inhibit microbial growth after all, just that it was not bacteria. It was all the fungus in the plate. We decided to isolate both funguses present in the plate and run further tests on AR and the other actinomycete AV against them. We dubbed the funguses H1 and H2. As we can see from the image in the previous post, AR was able to inhibit growth in both funguses, as opposed to AV and our control of course. That means that AR produces an antifungal substance. Now we look forward in doing further experiments to characterize H1 and H2 and to see if we can isolate the antifungal. 

Now it was a whole different story with AV. On the first test of antimicrobial activity of AV against our microbial targets, we observed something that looked like an inhibition zone in the S. aureus culture. Upon repetition of this assay we confirmed that indeed there was a 0.5 – 0.7 cm inhibition zone between S. aureus and the yellow pigment secreted by the actinomycete. Nothing against P. aeruginosa though. Therefore, it is safe to say that AV is secreting an antimicrobial agent against S. aureus, yet we would like to test the antimicrobial potential of AV against other gram positive-bacteria to verify its specificity.

PKS and NRPS Amplification

Amplicons acquired from the PCR were observed in 1% (w/v) agarose gel electrophoresis. Fragments amplified with K1F/MR6 primers have 1,200 – 1,400 bp (Figure 1). Fragments amplified with A3F/A7R primers have 700 – 800 bp (Not shown), encoding for conserved motifs in multidimeric peptides associated NRPS. Nonspecific amplifications were observed in both reactions.

Testing Isolated Actinomycetes Against Microbial Targets

Antimicrobial activity was observed from both actinomycetes towards distinct targets. Antifungal activity was observed from AR against fungal contaminants H1 and H2. The red pigment secreted by AR in solid media was not observed when it was cultured in liquid media. Antifungal activity was observed when solid media discs containing the red pigment were tested against fungal contaminants, as opposed to having the supernatants from the liquid cultures that were absent of the red pigment (Figure 2). No antifungal activity was observed from the second actinomycete, AV, given that it presented dearly identical fungal growth in both antibiograms. Antibacterial activity was observed from AV against Staphylococcus aureus, but not towards Pseudomonas aeruginosa, while AR had no apparent effect over either bacterium. As with AR’s pigment, AV secreted a yellow pigment in solid media only. Media disks containing the yellow pigment were effective against S. aureus presenting an inhibition zone of approximately 0.5 cm.

In the Near Future

We expect to use additional whole-cell techniques in order to detect antimicrobial activity from cultivable microbes. Also, we intend to verify whether the antimicrobial substances produced by AR and AV are novel.

1. Amplified PKS I and PKS II genes from last semesters’ antimicrobial agent-producing microbes.

2. Executed at least three more antibiograms (antimicrobial activity detection assays) to ensure reproducibility.

3. Determine the level of susceptibility of target specimens against the antimicrobial agent-producing microbes.

So far, we have amplified PKS I-encoding genes from two distinct soil samples. Furthermore, we expect to finish amplifying other PKS-encoding genes by the end of this month. This amplification process is carried-out by polymerase chain reactions (PCRs) aiming to screen large genomic libraries for potential antimicrobial activity.

Last month however, the objectives changed with the addition of two actinomycetes isolated by Vanessa Cardona, a master student from Dr. Ríos-Velázquez’s Lab. One of these actinomycetes has already shown antimicrobial activity against a common fungal contamination in the laboratory. While the other, presents dual pigmentation. Currently, we are assaying the antimicrobial potential of both actinomycetes, via modified Kirby-Bauer antibiotic activity tests. 

        Informative and path-changing is the best description I can think of for my first research experience outside of Puerto Rico. After a semesters’ worth of biotechnological research at the B 266 laboratory at the University of Puerto Rico – Mayagüez Campus, I had the opportunity of working in a biomedical engineering research laboratory at Yale University this summer. The research project I worked in focused on testing nanoparticle drug delivery systems as a means to treat immunological disorders in vitro. I learned a variety of techniques, all of which greatly differ with what I do in my research project at Puerto Rico, but I had a great time and made new friends. I learned methods such as cell culture harvesting, solution chemistry, nanoparticle synthesis and characterization, antibody staining, and immunological assays, among others.  Overall, the research experience was great and I am very glad and ready to start to work back at B 266, Laboratory of Microbial Biotechnology and Bioprospecting Research.

One of the greatest obstacles I’ve had to deal with in the research project is to balance the time in the lab with the time I have to invest in studying, eating and sleeping. Sacrificing many pleasures of daily life is just an example of how much time the research project can take, unless it is sufficiently organized. I intend to solve this problem by organizing my research activities as well as possible, and consulting the order I have chosen with my research mentor, when possible.

During the next semester I expect to be considerably more organized and keep up the hard work with a happy face and a cup of coffee.

                     In order to complete the blog entry requirements, I was assigned to visit the blogs of Yanira Marrero Rodríguez, Sheila M. González, and Laura Almodóvar. Visiting the blogs of these three students reminds me that biotechnological research is as interdisciplinary and diverse as science itself. Yanira, for example, is working in bioprocess engineering and her research goal is to develop an efficient method of converting biomass into ethanol, as means to fuel our everyday machinery. Contrary to Yanira’s alternative fuel development goal, Sheila is working in the ecological and molecular characterization of Cryptococcus neoformans in Puerto Rico, particularly focused in understanding the pathogenic routes of C. neoformans. At a difference from both Yanira’s and Sheila’s research, Laura is working in developing methods to remove heavy metals from plants in Adjuntas and Mayagüez, Puerto Rico. I also learned that although Sheila’s research appears to be rather local, the applications of her discoveries can affect crop growth and irrigation methods worldwide.

                 Almost three months have passed since I began working in B-266 Research Laboratory of Biotechnology and Bioprospecting. So far, I have learned a variety of molecular techniques that range from DNA electrophoresis, to preparation of Polymerase Chain Reaction (PCR) cocktails. Electrophoresis is a commonly employed molecular protocol that focuses in separating different macromolecules such as proteins and DNA. This separation technique is achieved thanks to the mobility of ions in an electric field (Physics!). Similarly, PCR is a commonly employed molecular technique, but instead of separating DNA or proteins, it amplifies nucleic acid fragments. Typical PCR uses specific cocktails composed of enzymes, nuclease-free water, primers, and a DNA sample.