The first project that I evaluated was “Reactivity of Hydrogen Sulfide with Hemeproteins”. This proyect had a lot of work ahead, specially in the writing area. However the proyect has interesting in the fact that its one of the first times that I can combine kinetics and Biology  and not get wildly confused. Sits not my favorite topic, Molini did a good work on explaining his work. My only recommendation is that when he writes his bastract, he should be able to explain what is the importance of his project. A thumb up for him.

The second poster that I evaluated was ” Enantioselective Reduction of Prochiral B-Halogenated Aryl Ketones for Organic Synthesis of Chiral Oxetanes”. Now this one I liked very much. This one explained how this enantioselective reduction helped to produce a much specific organic synthesis product. She explained to me the importance of this reactions. This can provide a larger yield with the same raw material. I give Irisbel Guzman a two thumbs up on her poster and project, she also projected a positive energy upon her research

The third and last project that I had to evaluate was “Generation of GFP Variants using Error Prone PCR and Characterization in Escherichia coli.” Bingo. This one I loved. This is all about Molecular Biology and Bioluminescence . BOOM, this looks a lot like my project in perspective to the techniques that were beings used and their utilities. This project was about generating GFP variants that are brighter and/or more resistant to extreme surroundings. This is useful in a lot of aspects because this GFP is commonly used to detect patterns and other molecular techniques. This poster and project deserves a 2 thumbs up.

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

Results from the 16s rDNA amplicons :

By looking at these results I confirmed that the 16s rDNA reactions give me the DNA that I will need to work this semester with the restrictions enzymes and produce the elaborate results that I desire.

The next 4 pictures are the luxH amplicons of the 60 sample run:

The most important objectives for this semester are the following:

·                     Design the proper primers for the amplification of the luxAB region inside the lux gene. This region is responsible for the production of luciferase so in order to clone the whole gene, this region is of innermost importance.  After these primers are designed the next obstacle will be to find the most suiting parameters for a proper PCR. After this is done, the amplification of the other lux gene segments will be amplified following the same steps. As a reminder, the amplification of the different lux genes can be used for a proper molecular method of identification between bioluminescent bacteria amongst Puerto Rico’s coastlines.

·                   Perform ‘in vitro’ analyses using several programs that can help us decide the proper restriction enzymes that can undertake an ARDRA that discriminates amongst bioluminescent bacteria in Puerto Rico.

The work in this semester is the continuance of last semester’s experimental work.

Still, this semester, I will probably perform several experiments that will help us identify our samples with a molecular method that involves the primers that were designed on the last semester’s experiments.

I would say that my progress for technical objectives is at 4. The progress of designing many sets of primers (many successfully) and amplifiying the genes continuously is the proof of it. Ive also managed to set a few conclusions by myself and these are going to be the next objectives for my investigation.

      When I had all the sequences that I wanted to study, I used another program to align them and therefore design the primer that I was going to use to amplify the area of the lux gene that interests or matters to the research.

I would say that my research deserves a 2 in progress because my first and one of the most important objective is basically covered and now i will proceed to verify if the method proposed works (which is my second objective: To do the tests in vivo). In the next few weeks I will inform on how things went out with the new set of primers.

I’ve encountered a few problems working with the programs in the design of primers, but these helped me to understand the concept completely and now I feel really comfortable on designing these.

See you soon.

The first meetings have taken place. Security measures and other important rules have been discussed in these meetings and this week I will resume my work on the bio-marker. In the summer I read various papers and basically understood what Im going to do on this semester.

A brief summary:
I will develop a molecular tool which will help identify the bio luminescent bacteria in Puerto Rico’s coasts.
I will do it using ARDRA (amplified ribosomal DNA restriction analysis) methods in which the LUX genes of the bacteria will be amplified.

The LUX gene is basically the same on most bio luminescent bacteria except for some few details.
These small but important differences will be the key for my molecular tool.
My first step will be to design a primer so I can amplify the LUX genes so I have a lot of DNA to work with.

The techniques I used in the last semester were done because by that time I did not have the knowledge of basic Microbiology and Genetics. Those techniques helped me understand the pourpuse of the research I will undertake this semester.

As a reminder, these next weeks I will be designing a primer that amplifies the LUX F gene.
This gene will help me distinguish among different bioluminescent bacteria.
Then I will be finding the proper restriction enzymes. But, lets go ONE step at a time.
I will inform you the success or failure that I get in some time.

STAY TUNED!!!!

-Diego Hernandez Aranda

This experience has been a powerful and enriching one. With all the responsibilities doubled and the tests getting harder and harder the level of study on this semester was increased exponentially. The amazing result is that I’ve made it trough. With the support of friends, colleagues and family, the work-study balance of this semester was succesfully taken on. Ive learned that there is to be a balance to the things I do if I want to keep on track and reach a far away horizon. I have learned, of course, the techniques I explained on the past blog entry and also the manner in which to look at a problem and solve it with logic and what I have done so far.

My goals for the next semester are to use the techniques I have learned so far so I can provide information and results on developing the proper bio-marker for the bacteria that I am currently studying. I will begin asDra methods to obtain this bio-tool and find out if its the proper one for these bioluminescent bacteria.

Extraction

In basic terms, this process that haves many steps basically isolates and cleans the DNA of the bacteria that that I wish to study.  Common chemicals used are Ethanol and Chloroform are basically used for the cleaning of fats and any other residue that isn’t DNA. And the Lysis Buffer solution fragmentates the bacteria so the DNA is poured out. RESULT: CLEAN DNA.

PCR

Is the step in which we make many replicates of the DNA that I am interested with. Useful because makes DNA more visible in electrophoresis and if some DNA is lost during procedures y have many copies to replace it. A common name in this procedure is the primer which marks the part of sequenced bases that I want the “coping” to start from. This step is commonly called the amplification of the DNA.

Electrophoresis

This is the step that brings out the “readable” data. Charge repulsion is the used rule for this method. The DNA carries a negative charge and so do electrons. The method basically is to carry a current so the DNA (repulsed by the negative charge) is repulsed. The heavier strands of DNA stay behind meanwhile the lighter travel farther on. This strands are divided if we “cut” the DNA with a restriction enzyme. If this is not done, a single and heavy strand of DNA will be the result. We can know how many base pairs does the strand have because a “ladder” ( its like a legend) tells us how many base pair we may have. It may sound like a very complicated process but, once you do it for the first time, it will be very easy to understand.

 BIO-BLOG COMMENTS:

Ive just submited the comments and I have seen that some are having the same problems that I am confronting. I notices that one of the students is going to do the same procedure that I am. I think that it is a good thing that the students are connecting because we can healp and support each other. Many nice blogs and I hpe to see many more.