Report on the semester’s objectives

The objectives for this semester are the following:

1. Amplify the luxH and 16S rRNA region from the whole bioluminescent bacteria collection at the lab.
2. Design primers (as many sets as needed) for the luxAB region of the lux gene in order to amplify it correctly.
3. Test the selected endonucleases for the ARDRA procedure.

This semesters objectives are at (3) status (50% complete).

The half of the whole collection has been amplified for the two regions and the other samples will be amplified in a very near future. Meanwhile the design for the luxAB primers has not been easy. The diferent sequences that are found in the NCBI page are not very homogeneous (the different sequences analyzed vary too much, which makes the design of primers a un-friendly procedure. I am about to suggest my mentor that we use the primers that have been already designed for this regoin instead of  designing a totally new one. Perhaps the “already designed” primers work with the samples that I am studying. As for the ARDRA problem, I have not recieved the enzymes (endonucleases) I need to get that part done. So that test is on a brief pause. I have not been to the lab a lot this last two weeks because I had many tests but this weekend I will post new results and my next plans.

The abstract for my investigetaion is the following:

           Marine bioluminescent bacteria are organisms that show luminescence phenomena by quorum sensing. A method that differentiates among the bacteria obtained in Puerto Rico’s coastlines is necessary because the large sample collection that the Island offers will produce an effective process that can be useful in the development of a bio-sensor. Using an amplified ribosomal DNA restriction analysis method, the 16S rDNA will be digested with endonucleases so that the different species of these bacteria can be identified. Another molecular method (lux gene method) of identification can be designed using lux gene fragments that differentiate between several genus and species (luxH and luxF are fragments that differentiate among Photobacterium and Vibriogenres of bioluminescent bacteria). Six isolated samples were amplified with the polymerase chain reaction method (PCR) in order to obtain the 16s rDNA region. As for the lux gene method, an in silico analysis was carried to design three primer sets for a luxFPCR and one set for the luxHPCR. It was seen that the primers for luxH produced a 700bp product with the six samples; in contrast, the luxF primers sets tested have not shown any positive results. The determination of an effective design of the luxF primer sets will be determined when these are tested with Photobacterium phosphoreum, a positive control. The next step will be to undergo digestions tests to the 16S rDNA with different endonucleases that determine restriction patterns that can be used as a method that distinguishes among bioluminescent bacteria in Puerto Rico.

I hope this helps.

Ill post again in a couple of days