Research progress

I have been working for over a year in this project (refer to GeMs to know more about it), and I can say that now I am seeing the light at the end of the tunnel. Right now we are at the first stages of our project, which is the construction of the Low Molecular Weight Metagenomic Libraries (LML). We were having some technical difficulties but gratefully we were capable of handling them. Once this step is completed, we will move on to the second stage of the project, which is “Screening”.  Our goal at this point is to construct over a hundred of libraries on this semester and sadly I have none, but not for so long

February 27,2008

It has been a month since I became member of the Biominds program. A month full of work and great experiences working with my team. My lab work team is composed of ten persons but I only work directly with one other person. As first, ten persons could be seen as a lot of persons to work with at a lab, but I can say that for me it has been a wonderful experience so far. Each one of us, working in the same project, but, with different perspectives and procedures, conforms an essential part needed for the project’s fulfillment.As my research progress regards, I am still having some technical difficulties I need to overcome. As you may know my research tries to access the metagenomics of the soil, specially by cloning techniques. This field involves the use of special techniques on areas such as genetic engineering and molecular biology, sciences, that are very sensitive to external factors. Because of this, I’ am experiencing problems at the molecular level, specially in the ligation of the DNA with the vector and with the DNA gene cleaning process. For some reason my plasmid does not enter the electro competent cells. I am already trying to identify the possible source of the problem. Right now I have done the tests needed to discard the vector and electro competent cells as the source of the problem. By this week I will make other tests in other to identify the possible problem.

March 28, 2008

The construction of Metagenomic Libraries involves many molecular techniques. Such techniques can be a little troublesome at first but once you know them are very easy to work with. (I can say that is not my case…yet!) Among such techniques are, DNA extraction from soil. We do the extractions with already made kits which consists of mixing and centrifugation of the kit solutions’ and the soil. It is an easy step. The only problem is that we need to be very cautious not to break to much the DNA. Another technique we use is the DNA digestion. First we identified with which restriction enzyme we want to cut the vector (in our case a BAC vector), then we make the proper calculations to cut the vector and DNA. After the digestion we proceed to take out the phosphate groups of the vector, ligate the DNA with the vector, and proceed with the electroporation of the conjugated vector into the competent cells.As part of our monthly report, I visited other three BioBlogs. From them I learned the wide variety of investigations undergraduates made in different areas. It is amazing However, even though our science interests are different we have the same desire of contributing to forge a better place to live. I am very confident that each one of us at Biominds will contribute in different ways to build it.

April 28, 2008

The end of the semester is near and with it, the end of a full 5 months of hard work. During this period I learned not only research techniques, but  also important knowledge concepts of the molecular work I am doing in the laboratory.

Among the techniques and concepts I learned during this semester in the laboratory are:

Molecular Biology

It has been the area were I learn the most. I learned how to extract environmental DNA, how to cut my DNA in order to obtain an approximation of the length I want, how to digest a vector, how to remove it phosphates groups after digestion, how to ligate the plasmid with the foreign DNA and how to insert it into a host for expression. In the conceptual basis I learned how to create my own digestion reactions using the manufacturers recommendations taking into account my DNA concentrations and final concentrations of the buffer and enzyme.

Microbiology

From this area I learned how to work under aseptic conditions. Our research involves a part where we need to inoculate bacteria and prepare media. Even though we use selective media (using an antibiotic for selection) if we are not careful enough we could contaminate our media with microorganism such as fungi or some resistant bacterial strain. (Been there)! Among the techniques I learned are: preparation of agar plates, inoculation of bacteria, and preparation of broth and solid media.

Team Work

One aspect I believed is the most important among the concepts I learned is teamwork. In real life situations no one works alone. The experience of working in teams of two people has taught me how to handle difficulties and situations with a lab partner. Also this teamwork experience will help me to be better prepare for my graduate studies if at some point I need to work with someone.

I believe learning is a process of cause and effect. In order to learn from an experience you need to overcome its barriers. For me this research experience  has been of no exception. Our research aims to isolate novel genes from metagenomic libraries but in order to do that we need to construct it first. This process has been very tedious. Molecular Biology is very sensitive to external factors and the complete process of creating a library involves Molecular Biology. At first we were having problems trying to isolate the vector from the bacteria having it, we could not determine the optimal enzyme amount for cutting the environmental DNA, we could not ligate the plasmid with the DNA and we could not obtain any bacterial transformant from the electroporation. In order to overcome this problems we did an intensive search on the literature in order to optimize the protocols we were using. After a lot of hard work and trying once and again, we finally obtain possible prospective clones .

With the clones created, during this time and next semester, we will first Identify if they are positive clones (that in fact the host contains the plasmid and the foreign DNA). Once identified, we will proceed  to screen the metagenomic libraries for novel active biologically molecules. Also we will still constructing more libraries for further screening.

August 25, 2008

Summer at UW-Madison

During the months of May through August, I was accepted to perform undergraduate research at the University of Wisconsin-Madison Integrated Biological Sciences Research Program (IBS-SRP) with the Cellular and Molecular Biology sub-group. For my surprise I worked in the Chemistry Department in the Biophysics training program under the mentoring of Kem Winter, Benjamin Bratton and Dr. James Weisshaar as principal investigator.

You may think about the non-correlation between cell bio and chemistry but I was totally amazed when I knew the topic of my summer research. My project was to describe how osmotic effects changes the diffusion of green fluorescent protein (GFP) in the periplasm of Escherichia coli. The main purpose of this study was to grasp a better understanding of the sub cellular dynamics of the periplasm of E.coli and the physical changes due to osmotic effects. This type of experiment will aid us to determine many thermodynamic properties of molecules of the same size and shape as GFP.

The experience was amazing. This type of project allowed me to make a connection between my engineering courses as algorithm and unit operations, my chemistry courses as physical chemistry, my biology courses and my statistical courses, which sometimes can be really hard when someone is studying an interdisciplinary subject as Biotechnology.

The knowledge acquired in this summer program was really significant. Between my tasks as an undergraduate intern were to grow bacterial colonies, be able to handle optics, lasers and fluorescent microscopes, instruments used to determine the diffusion and computer programs such as IDL and Metamorph used to analyze the data.

This summer gave me a better insight of what should I expect in graduate school. The fact that you are alone working in a project new to you with new people helps you to develop characteristics that in a classroom are impossible to acquire.

It was really a great summer!

Objectives proposed for this semester 2008-2009

During the previous semester our main objective was to develop small DNA insert metagenomic libraries using a BAC vector. This semester we plan to continue the BAC library construction and in top of that, at the end of the semester our aim is to:

• Be able to generate a BAC library of approximately 30,000 clones
• Be able to generate a fosmid library of approximately 100,000 clones
• Screen the libraries for the presence of antimicrobial, lipolytic and agarolytic activity among the clones.
• Characterize each potential gene conferring the activity.

This work pretends to be a continuation of the previous work done in the past semester.

The past semester we did not construct any library. We had several issues regarding DNA concentration, and ligation problems. As part of it we spend most of the time optimizing the protocol and trying to figure out the cause of the problem. This semester we plan to construct both libraries.

One of our objectives for this semester is to construct a fosmid library. The main difference between this library and the BAC library is the method used for the DNA insertion. On the fosmid library the DNA is inserted via a virus packaging meaning that the virus with the DNA infects the bacteria cell. On the contrary the DNA in the BAC library is inserted by a ligation reaction between the DNA and vector. Then the bacterium is transform with the plasmid via chemical transformation or electroporation. Those are our major aims for this semester.

This semester looks very promising.

September 26, 2008

Among the several techniques employed in the construction of metagenomic libraries, a common one used to identify and study the community’s phylogenetic is the construction of 16 S rDNA. 16 S rDNA is a gene that sequence for one of the prokaryotes ribosomal unit. This gene is conserved among organisms, which provides an excellent marker to differentiate among many. This technique is similar to constructing a library. The only different step is that after DNA extraction from an environmental sample or from a previous library, we amplified the 16 S rDNA by PCR, and only this fragment is used for cloning procedures. Our DNA is going to be extracted from the previous constructed libraries.

Regarding to my progress I believe I am at level 2. Up to this date I have completed 25% of my objectives posted on August 25, since from 4 objectives I have been able to accomplished at least one. We have successfully cloned libraries from around 25 to 40 kb from two completely different environments. One library was constructed from a rainy forest and the other one from a dry forest.

Working in this project for almost two years now, taught me that science does not always goes as planned. In my case, I have been struggle for creating a library, but now I can say that I finally overcame that problem. Now, the next difficulties in the way, is going to be able to select the right activity for screening, or to isolate a clone with a specific activity. This is because in order to have activity, the clone must have its gene, meaning its DNA, which finally means that, it was cloned altogether. Since we cut our DNA randomly to eliminate bias we do not know if in our cloning are complete clusters of genes. That is what are we going to find out. To solve this, is why we are cloning huge fragments of DNA, to increase our probabilities of cloning complete genes and not fragments.

October 26, 2008

Up to this point, regarding to my research progress I believe I am still at level two. With the library made, my next step is to search for antifungal activity among my clones. This, means that I need to search for a particular clone that when grown with Saccharomyces cerevisiae inhibits it’s growth. This is done by patting the library, by doing this I will have the clones growing as separate colonies in the plate. After three days I add to the plates a Sacharomyces suspension in soft agar. If a particular clone produced some substance that inhibits the Sacharomyces growth I will see a clear halo surrounding the colony. I started the procedure but unfortunately  I have not found any clone yet.

Antifungal substances, as the word suggests, are substances that inhibits fungal growth. My main focus is to be able to isolate novel substances with this peculiar characteristic from the library. The isolation of a novel molecule like this does will have a great impact on the scientific community as well as in our society. If the substance is produce it must be because of a particular gene. Characterizing this gene will aid us in the understanding of several bacterial mechanisms. For example how they synthesize the molecule, how is exported outside the cell if exported etc. Saccharomyces is used as a model subject in this experiment. The next step after the isolation of the molecule will be to see if it also inhibits the growth of pathogens fungus like Cryptococcus neoformans etc.

November 25, 2008

The first 2008-2009 semester is over, and with it a year of ups and downs in our research. Our main research goal is to obtain metagenomic clones for the further study and screening of novel biological active molecules. During this semester we were able to finally construct those forest soil clones. The two soils under study were “El Yunque” and Guanica’s Dry Forest. In total we obtained a library of 795,830 clones. 14,631 clones were from “El Yunque” and 781,199 clones were from Guanica’s Dry Forest. In the photo to the left you can see a restriction digest analysis of some of the dry forest clones. Here you can see the DNA fragments within the vector of each individual clone. The library was constructed using the fosmid vector pEpiFOS™-5. With this, we complete one aim of the research which was to construct the library. With the library constructed we proceed now to actually screen it to see if we clone for any chance any fragments coding for active biological molecules. Our screening focuses on the isolation and characterization of antifungal activity, as stated before. To date 5,600 clones, from the “El Yunque” library, had been screened and no antifungal activity had been observed for the moment. Our plans are still the same, screen the complete library to see if an antifungal clone can be selected. For the moment this next semester we will start other screening, in addition to the one described above. We will start looking for clones with lipases. This semester has been one of hard work but really productive. Now that the library is created we can focus all our time to isolate a clone with a possible activity.

January 27, 2009

A new year starts and with it a new semester with new goals. In my case some of the goals for this semester experiments are pretty much the same. By the end of this semester we hope to be able to:

• at least isolate one colony with antifungal activity among the clones from the library created. We have been screening for the past semester but we have not been lucky.
• because of the complexity of the screening procedure to identify a candidate we will be designing new and improved methods to make the selection process faster.
• we will identify other important biomolecules in order to search their presence in both of the created libraries

In this past semester we were able to construct two libraries from two different forest. Even though our plans were to isolate also a candidate with a positive activity the time was not enough to fulfill both objectives: the construction of the library and the isolation of the clone. Now with the library created I can focus all my efforts on trying new techniques in order to find a clone with an antifungal activity. Therefore, both semesters are related since this semester I will continue with the second part of the project: lookout for an activity in the library.

I need to mention that the screening was started this past semester, but we did not found any among the 5,600 clones screened. Since we need to screen an enormous number of clones we want to identify quicker methods for the detection of the antifungal activity. This will be our main objective for this semester.

February 28, 2008

Up to this month I had achieve a progress of 1. Looking for antifungal activity has been more challenging than expected. This arise as a result of the total number of clones I need to screen and because I am looking for the activity one by one, colony per colony. Just to give you an insight of the dimensions of the project. First I inoculate the library in 1 mL of LB for two hours. The I make a 1/10 dilution in a total volume of 1 mL, therefore from my first culture I can make 10 dilutions of 1 mL. From this dilutions I proceed to plate 100 uL meaning 10 plates/dilutions. Having a total of 10 dilutions it transfers to 100 plates in just one screening. I am posting my abstract so you can have a better picture. However I am pretty confident I will be able to find something.

A previous lab co-worker was using molecular techniques to see if it was possible to amplify PKS, genes responsible for the production of antifungal molecules. He was able to make an amplification, meaning that indeed there are known PKS genes in the library. However with my approach I am trying to go a little further; to identify a novel PKS gene.

Screening for Antifungal Activity in Forest Soils in Puerto Rico using culture independent and functional genomics approaches

Studies have demonstrated that standard cultivable methods fail to access a representative amount of the microbes available from a microbial environment population since they only obtain a 1% of the whole population. This leaves behind the other 99% of the microbes, which can represent organisms with possible phenotypes never described in scientific literature. To access this diversity we created two fosmid libraries of soil genomic DNA from El Yunque National Rain Forest, and Guanica’s Dry Forest. The total library, from both forests consisted of 795,830 clones from which 781,199 clones were from Guanica’s Dry Forest and 14,631 clones from El Yunque. Among the several novel biomolecules searched, novel antifungal molecules able to inhibit the growth of Sacharomyces cerevisiae are of main interest. Because of the high number of plants and human pathogenic fungi already resistant to several commercial antifungal components, this novel method for the identification of new antifungal components promises to be a viable alternative for controlling such diseases. The library stock pools were grown for 2 hrs and serially diluted until obtaining 600-800 clones/plates. Once the dilution factor was obtained the library was cultured in LB agar supplemented with chloramphenicol for 3 days at 37°C. The precultured clones were overlaid with 5 mL of LB soft agar containing a S. cerevisiae suspension in Yeast Potato Dextrose broth adjusted to an optical density of 0.2-0.3 Abs. The plates were incubated overnight at 30°C and scored for activity looking for inhibition zones in the S. cerevisiae lawn. To date 5,850 clones from El Yunque Rain Forest have been screened and no antifungal activity has been detected under the current experimental conditions. With the isolation of a positive clone from the library we will proceed to characterize its gene cluster and identify if the antifungal component also inhibits the growth of other pathogenic fungi.

April 29, 2009

On March 21 Biominds celebrated the Biominds Research Day. This research day was a symposium where each one of the biominds students had the opportunity to present their research done so far during these two years. As part of the experience, I had the chance to visit three different posters with the objective of getting more knowledge in their research areas. Because of time issues, I only had the opportunity to interview only one student, however I was able to extract some information of the other two students work by reading their abstracts. The name of the students are Jessenia Yaris Laguna, Rocio Rivera, and Sirena Montalvo

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Jessenia Yaris – “Identifying Genes potentially regulated in the hippocampus by DNA recombination”

Jessenia is a UPR-Rio Piedras student. She is trying to elucidate the molecular mechanisms by which long term memory forms in the hippocampus. According to her hypothesis, long term memory in the hippocampus forms by a DNA recombination process that involves the rearrangement of specific genes. Considering many diseases that involves neuronal damage such as Alzheimer, where long term memory is affected, this type of research can serve as a possible treatment for preventing memory damage. Thus, having a great impact in the scientific community  if the mechanism gets described.

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Rocio Rivera – “Generation and Screening of Metagenomic Libraries from a tropical forest in Puerto Rico: Searching for novel enzymatic capabilities”

Rocio is a student from UPR-Mayagüez. Rocio’s research topic is also Metagenomics. She is trying to find novel genes encoding for amylases, 4-hydroxybutyrate dehydrogenase, and poly(3-hydroxyalkanoate), using independent culture techniques. This allows her to investigate the presence of these genes in organisms that do not grow under standard laboratory conditions, thus increasing her chances to find them. The importance of this project relies in the fact that she can use the same method to look for other enzymes used in industrial processes. This way, will satisfy the need for novel enzymes needed for new biotechnological processes.

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Sirena Montalvo- “Microbial Community associated to the bovine udder”

Se is also a student from UPR-Mayagüez. Her work is in the area of microbiology. Mastitis is a common disease among cows. It consist of a lesion infected with pathogenic bacteria in the cow’s udder skin. Normally it can be treated with antibiotics, however it represents a high cost to the milking industry. In order to try to reduce treatment costs, she is trying the possibility to treat the infected cows with natural microbial communities. She will determine the microbial communities on infected and non infected cows in order to see if there is a difference in natural microbial flora and if there is, she will treat the infected one with the non infected microbial flora. Reducing costs will benefit not only the industry but also the consumer because it will mean a price reduction on the milk.