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.

 

 

At the beginning of this semester, my professor and I planned that by the end of this semester I should have:

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. 

One of the techniques I have learned in the Lab so far is PCR-based genetic screening of soil DNA samples. I will use this technique to amplify PKS I-II encoding genes. If these PKS genes are amplified, then I will proceed to isolate and those microorganisms that produce PKSs. Up to this date, I believe my progress can be rated by a 3 (around 50% done), because I still need to isolate potential PKS-producing microorganisms. This progress is mainly due to my major obstacle: Too many exams, reports, and presentations, and very small amounts of time to work in the lab. Hopefully, this will be over by the end of this week. As I won’t have tests for two weeks or so.

        

        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.