— Written by Vivian Lee

Through observing the presence of the 16s ribosomal unit from the bacterial samples that were cultured on various agar plates, the next step of identification was to get a closer look into the fingerprint. By using RFLP, which stands for Restriction Fragment Length Polymorphism, more distinctive banding patterns from the samples were differentiated through various enzymes. In RFLP, enzymes play an important role, as they break the DNA samples into small pieces at various restriction sites, they make each fragment distinguishable from one another on an agarose gel through gel electrophoresis. The enzymes that were used include HaeIII, DdeI, HinfI, HhaI, and RSAI. As the identification continued, the samples that underwent both a PCR and the RFLP were sent off for sequencing.

Figure 1: Through Gel electrophoresis, Polymerase Chain Reaction (PCR) results displayed the presence of the 16s ribosomal unit in multiple sea star bacterial samples. The presence of the 16s ribosomal unit is identifiable at around 540bp.

So what does sequencing my samples mean? Sequencing the bacterial DNA samples allowed me to identify which strain of bacteria matched the DNA of our sample. Through this recognition, results have shown that there is a presence of Vibrio, as well as other bacteria, including the Pseudoalteromonas. Through these results, what can be said about the role of Vibrio and its effect on Sea Stars is still being questioned.  The identification of Vibrio puts us one step closer to understanding that among the Sea Stars that were sampled, sick stars had Vibrio present. There are still numerous questions that need to be answered and various parameters that need to be taken into consideration. Are there are Sea Stars in intertidal that are currently alive and healthy, and if so, how are these survivors different amongst different species? Genetically, are these survivors going to advance to defend themselves in the future from another possible disease outbreak? The next approach to isolating other strains of bacteria, not only Vibrio may lead to another possible culprit that may be responsible for the deaths of these stars.

Figure 2: Gel results from a RFLP. Gel displays four different bacterial samples that experinced RFLP using five different enzymes (HaeIII, DdeI, HhaI, RsaI, HinfI, Sau3AI)