about our poster day…

During the annual BioMINDS poster day I had the opportunity to visit three posters with research results from work done by my fellow BioMINDS scholars.  The first one I visited was done by Isabelita Martínez from the UPR Mayaguez campus.  The title of her project was “Isolation of Important Clinic Yeasts on Water Treatment Plants and Puerto Rican Forests”.  Ramonita’s poster was very complete and she greeted us with a smile on her face, even though her three evaluators visited her at the same time.  She seemed very in control of the situation; she answered every single question we gave her.

 

The second poster I visited was titled “Isolation and Characterization of Puerto Rican Soils Bioprospects Resistant to Metals”.  Gladiany’s project, also from the Mayaguez campus, was very interesting because it’s related to astrobiology, which I think is a very exciting and fresh field of study.  I particularly like this poster because it relates to my project.  It was focused on metal resistance while mine was focused on antibiotic resistance.  

 

My third and final poster evaluation was on “Photo degradation of Promazine in PBS/7.4”.  This poster presentation was given by two of my fellow owls from my campus, Humacao.  It was really interesting to see what other students in my campus were doing, especially things totally different from what I’m experience in.

 

I was amazed by how fast this past three semesters had gone by.  We have made so much progress… 

wrapping-it-up…

To this day we have about 80% of our work done.  We are still working on sequencing our clones.  After that, it’s all about bioinformatics… in order to interpret our results.  I hope that we can have the sequencing results in order to include them in my poster for the BioMinds Symposium!

 

Search for antibiotic resistance genes from Metagenomic Libraries Derived from Microbial Mats at the Cabo Rojo Salterns

Casanova Angel,^1* Robert Rabelo,¹ Lilliam Casillas,¹  Karen Cloud-Hansen², Heather K. Green² and Carlos Rios-Velazquez ³

¹University of PR-Humacao, ²University of Wisconsin-Madison, ³University of Puerto Rico-Mayaguez

The Cabo Rojo Salterns located at the Southwestern region of Puerto Rico posses a series of salt ponds surrounded by microbial mats where microorganisms are subjected to extreme conditions. According to prior phylogenetic studies, most microorganisms in the mats belong to novel unidentified groups which make the mats excellent locations for the construction of metagenomic libraries.  Direct cloning of environmental DNA from the mats might have great potential for identifying new genes and gene products for biotechnological purposes particularly those conferring resistance to antibiotics. To determine if novel antibiotic resistance genes can be found from the mats we constructed a metagenomic library from the mats of the Candelaria lagoon. Genomic DNA was extracted from the Candelaria microbial mats using an indirect method, and a fosmid library of approximately 33,000 clones was generated in Escherichia coli as a host. The clones in the fosmid library carried inserts of more than 20Kbp. The library was then screened for antibiotic resistance genes using agar plates supplemented with Carbenicillin and Gentamycin. From the initial screening, seven positive clones were chosen, tested for the presence of an insert, and reconfirmed for the antibiotic resistance.  To date the preliminary sequencing analysis conducted revealed genes of unrelated functions within the 20Kbp insert. Sequencing efforts are currently underway to determine the gene responsible for this activity and further determine if novel mechanisms for antibiotic resistance can be reported from these clones isolated from the Cabo Rojo salterns microbial mats.

about to wrap-it-up!

Last semester we encountered many difficulties trying to optimize our culture method. Due to that inconvenience, this Christmas vacation I engaged in another project in order to have a something to present in our poster day. I must I confess that we have achieve a great deal of progress this past month. We were even able to have some preliminary results and submitted an abstract for the ASM Conference, which will be held in Philadelphia this year. That being said, for this semester my primary goal is to sequence DNA we have extracted from clones that have a plasmid insert from microbial mats at the Cabo Rojo Salterns and find out which are the genes confer antibiotic resistance. Moreover, efforts are currently underway to determine the gene responsible for this activity and further determine if novel mechanisms for antibiotic resistance can be reported from these clones isolated from the Cabo Rojo salterns microbial mats.

This project is very similar to the one I was previously working on. I am still working a metagenomic library from the Cabo Rojo Salterns, but this time I am looking for antibiotic resistance instead of enzymes. Direct cloning of environmental DNA from the mats might have great potential for identifying new genes and gene products for biotechnological purposes particularly those conferring resistance to antibiotics.

We constructed a metagenomic library from the mats of the Candelaria lagoon. Genomic DNA was extracted from the Candelaria microbial mats using an indirect method, and a fosmid library of approximately 33,000 clones was generated in Escherichia coli as a host.

about results…

We have been working on identifying clones, from a genomic library of the Microbial Observatory in Cabo Rojo, that are capable of using complex sugars as their sole carbon source. We have isolated DNA from an environmental sample of the Microbial Observatory, cloned a DNA segment of 30 kb into a vector (plasmid), and transformed the clones into a host bacterium, Escherichia coli. My work has been to screen the resulting transformants in order to find clones which posess the enzymes necessary to degrade EPS.

Microbial degradation of EPS was assesses using Biolog to measure the optical density of the medium in which the clones where being culture. Optical density refers to the measure of the amount of light absorbed by a suspension of bacterial cells or a solution of an organic molecule. We are using these values to measure turbidity, which in turn helps us estimate the concentration of polysaccharide molecules in our medium.

As a culture medium we use M9 minimal salts, 5x. This medium is commonly used for the propagation of E. coli and for plasmid amplification.

To assess the degradation of EPS we used Biolog. Optical density values where used to measure turbidity and estimate the concentration of polysaccharide molecules in the medium. O.D. was measured at two different wavelengths, 750 nm and 590 nm.

We were able to measure the optical density in 376 clones. Optical density, in the medium, decreased after a considerable period of incubation. This is due to the decrease in the concentration of polysaccharide molecules.

We have now prepared our medium with a lower concentration of sugars. We think this will help us assess the degradation of EPS in a shorter period of time. Our ultimate research goal is to discover new genes and enzymes, with Biotechnological and Biomedical applications.

about research contributions…

We have managed to obtain the correct concentration of bacterium in order to assess our objectives. We have cultured our clones in LB agar, inoculated them into microtiter dishes with LB broth. Finally, we are now growing them in M9 minimal salts medium with azo-barley-glucan (sugar) as a carbon source. In order to assess if the bacterium are using this polysaccharid, we are measuring the optical density using BioLog. That being said, it is safe to say that we have reach approximately 50% of our research goals (3 out of 5 according to the scale provided).

Moreover, I think it would be great to know and confirm that this complex sugar is being used by the bacterium as a carbon source.

Techniques… techniques…

To this day I have only been working with serial dilutions, wich is coveted skill for every microbiologist.  We seem to be experiencing some problems with our metagenomic library pools.  The bacterium are not growing as they should.  We have been trying to solve this problem by adjusting the media’s pH value and preparing different concentrations of bacterium.  On the other hand, we have prepared the broth in which we will grow our bacteria (LB) to keep them as stock.  That being said, we have only achieved 10% of the propose objectives.

New semester, new screening…

Here I am, again… and I have a story to tell!

This summer I had the chance to participate in a microbiology internship in the University of Wisconsin at Madison. I worked in Dr. Cameron Currie’s lab with bacterial cellulose degraders in leaf-cutter ant colonies. Leaf-cutter ants (LCA) are a unique group of insects that have evolved an advanced agricultural system based on a quadripartite symbiosis with the ant-fungus mutualism at its core. LCA actively cultivate fungal gardens, which they use as their food source, by providing the fungal cultivars with fresh plant material. In addition to the ant-fungus mutualism, LCA engage in symbioses with bacteria as well. We proposed that one role played by the bacteria in the ant-fungus mutualism is to facilitate the breakdown of cellulose. To test this hypothesis I had to apply techniques I alredy new from my research here in the UPR-H. In contrast, I learned new techniques that I know will be helpful in the research I will engage in this semester.

Last semester we didn’t have success with our urease screening and we decided to engage in an other screening, although we will work on our previous on at a later time. Accordingly, we will continue on screening the Cabo Rojo Microbial Salterns metagenomic library, but this time we will screen for genes capable of degrading soluble and insoluble sugars. That being said, we hope to find genes capable of degrading Dextran and Azo-barley Glucan which are the sugars we will be using. The techniques we will use for this screening are somewhat similar, at its core, to our previous one, but differ in complexity. We will use new techniques that will help us optimize our protocols and increase the number of bacterial cells submitted to the assay. For example, since there are 30,000 clones in each library tube, using micro titer dishes will be a very helpful tool.

We have big expectations for this semester, which promises to be a very exciting one. Stay tuned for future updates!

four months later… conclusions…

During the last four months I have learned many things, from preparing media to screening methods.  Thanks to that, I am more than excited to take my first microbiology course next semester.  I never thought I could be more passionate about microbiology than I already was.

Nevertheless, there has also been some barriers.  At first, the screening method I was using to find E coli. clones that hydrolyse urea was not working.  The clones were not growing at all, I presume this was due to the stress the media provided and the small amount of bacteria the screening required.  To overcome that barrier, I recently began to use another screening method, one that required a higher quantity of bacteria.  Hence, the clones began to grow as they normally do in other media.

My main objective and expectation for the next semester is to culture a higher number of clones and hopefully find one that hydrolyzes urea.  In conclusion, this has been a wonderful experience to the extent that it has motivated me to participate in a summer internship to continue learning methods and research techniques. 

three months and learning…

This experience so far has been a great learning tool because even though I have not taken a microbiology course, yet, I have learned a couple of techniques used to study microorganisms.

Among these techniques are:  aseptic techniques, enrichment, media preparation, dilutions, biochemical tests, different screening methods… All of these techniques are extremely important because all of them combined are necessary to conduct my research.  They help me to prevent contamination of my work and reduce the amount of bacteria in a specific area in order for me to study separate colonies.  Moreover, with biochemical tests I am able to evaluate the products of gene function, in my case the enzyme urease.

Finally, visiting my fellow BioMinds scholars blogs widen my perception and knowledge about the great deal of things that can and are being done in science.  It’s nice to know that all of us are really excited about our research projects.

Research… two months later…

I have had a great experience working with my lab mates. It is very comforting knowing that there are other people working toward my same goals. Hence, this gives me a sense of companionship, confidence, and the “umps” needed to work even harder because there are others who are counting on me and my work. All the hours and experience sharing have made us more than lab mates, we are friends.

I love it when we have a “full house”; all the team members are working at the same time. Even though the space may be crowded each one of us respects the space and work of the others. I think the most challenging part of working with friends is trying not to get distracted. ^_^ Nevertheless, that is also the most rewarding part because not only am I working on something I really like, but I’m working in a light and comfortable atmosphere. These are the things that make research an awesome experience: love for the things you are doing and working in a great environment.