Phylogenomic - Based Case Study : An Undergraduate Research Pedagogy at Kentucky

Currently the undergraduate biology curriculum at 2 and 4-year colleges especially in Historically Black Colleges and Universities (HBCUs) is undergoing a facelift by implementing a range of recommendations of committees on education. This has produced a demand to educate and train the current and future generation of undergraduates as scientists capable to perform modern biological research that is heavily dependent on genomic training, such as phylogenomics. It is our observation that the HBCUs may not have a strong infrastructure enough to meet this challenge without the development of vigorous research-based learning programs to complement the traditional lecture-only based configuration. In this case study, the first author undertook the objective to facilitate an undergraduate genomic research-pedagogy at the biology department in Kentucky State University, the only HBCU in the Commonwealth of Kentucky. The wet-lab activities in this NSF funded research project include molecular microbiology training in culturing bacteria, isolating DNA, performing PCR, cloning amplicons and analyzing the sequence data. The associated case-study subject was assessed in gaining of wet-lab skills, knowledge, application of protocols, analysis of results and impact on career choice. The results reveal an enhanced student confidence in handling of molecular techniques and positive admiration towards bioinformatics. It suggests that the wet-lab based research pedagogy could play a role in retention of students in biology. Although this case-study subject has appreciated the value of the phylogenomic approaches, a substantial and continual support is required to retain student interest especially institution like this HBCU. Keywords: Genomic research-pedagogy, Case Study, Kentucky State University Introduction According to the “Committee on Undergraduate Biology Education to Prepare Research Scientists for the 21 century”, (CUBE, 2003), it is recommended to undertake sweeping changes in biology curriculum and education at undergraduate level especially introducing computer based course improvement and curricular adaptation. The exploding advancement in bioinformatics forced many institutions to incorporate computing information across many discipline especially in biology (Campbell, 2003). Research-based phylogenetic learning, which incorporates genomic studies, is more effective (Suchman, et al., 2001) in biology especially in microbiological sciences. In recent years, genomic information on microbes has tremendously increased since more and more students were involved in the genomic studies (Chan, 2005; Campbell, 2003). Though microbial genomic analyses play a major role in the genomic revolution, many existing hypothetical sequences are not yet annotated. A couple of years ago, it was reported that only 3 % of the sequences are not labeled as hypothetical, which means the rest of those floating sequences are still to be annotated (Brown & Sjolander, 2006). There may be multiple reasons, such as poor bioinformatics training, lack of knowledge or lack of adequate categorization of microbial groups (like medical microbes, agriculture microbes), or untapped bacterial genomics etc. Besides it was reported Kentucky Journal of Excellence in College Teaching and Learning 29 
 earlier that there is lack of sequence identifiers (Brown & Sjolander, 2006). No matter what blocks the flow it is a fact that there are vast hypothetical sequences need immediate attention. There fore, custom designed development of computer-based training activities in phylogenetics is essential and it should start at high school level (Marbach, Rotbain & Stavy, 2008). Minority students’ participation in annotation process particularly in phylogenomics using bioinformatics needs significant attention, especially in 2-and 4year colleges. Even though as little as 3 % of the annotation was carried out using wetlabs about 97 % of sequences, which were annotated in the public databases, were done based on electronic evidences (Brown & Sjolander, 2006). Training undergraduate students in this direction using this emerging field with wet-lab activities helps in their learning process (Jurkowski, Reid & Labov, 2007) and supports their retention in Biology. However, the need to transform undergraduate learning is challenging (Labov, 2004) especially when considering dry and wet-lab training as it requires an indepth radical approach both in trainee and trainer. It needs equipments, updated technology, and service-mined human resources. Lack of bioinformatics training at their undergraduate level in many colleges leaves a significant gap in student learning process even to understand the basics of a phylogenetic tree (Meir et al., 2007). Some Historically Black Colleges and Universities (HBCUs) are lagging behind in this frontier-field due to lack of funding (Holtzclaw, et al., 2006) and dearth of infrastructures. As a result, the students are not well represented in computational fields of sciences especially in graduate studies. In some cases, the undergraduates are lagging behind even in basic biology education (Jurkowski, Reid & Labov, 2007). It is therefore important to train undergraduates, especially minority students, to improve their participation in phylogenomic-based studies. Undergraduate research experience has been shown to have a positive effect on career choices, development of independent thinking and enhanced participation in the courses that followed after the research experience (Lopatto, 2004). Such research experience boosts their grade point average (GPA) higher and even helps them to pursue PhD degree in science and engineering. With this objective in mind, many attempts were made in the past to introduce researchoriented courses at the 4-year colleges and universities. Induction of bioinformatics into the undergraduate curriculum has been attempted elsewhere successfully as reported earlier (Campbell, 2003; Chan, 2005). However in the absence of such curriculum at Kentucky State University, which is a historically black, liberal arts and 1890 Land-Grant higher learning institute, an attempt was made to induce the research interest in the Phylogenomics with the support of a National Science Foundation grant. In this article, we are exposing the wet-lab methodology of phylogenomic inference of Glutamate Synthase of a new soil isolate Arthrobacter nicotianae in an attempt to bring out the research pedagogy by using a case study model towards the phylogenomics field. Materials and Methods Conventional Microbiology Activities The approach was to assign a basic genomic-based research project to be carried out in the wet-lab for a semester period. The case-study subject (CSS) was an undergraduate minority student who has no prior knowledge in this field. The theory and experimental methods to be applied was instructed in the form of written protocol as well as in association with a competent Kentucky Journal of Excellence in College Teaching and Learning 30 
 researcher. In this single-subject case study, newly isolated Arthrobacter nicotianae strain PR was used throughout the study. The identification of this soil bacterium had been performed and confirmed using 16sRNA with high resolution automated microbial identification techniques through rRNA gene sequences, hybridization probes and molecular profiles (Accugenix, DE). The CSS grew the bacterium in Nutrient Broth (10mL) at 30°C, by picking a single colony from a Nutrient Agar stock-plate prepared earlier using a streak-plate technique. Genomic DNA was isolated using DNAzol direct kit (Catalog # DN 131, Molecular Research Center, Inc. OH) by mixing 1-10uL or 1-10mg of solid sample with 0.1mL of DNAzol Direct. The lysate was thoroughly mixed and a 2-5μL aliquot transferred directly into 20-50μL of PCR mix. PCR-based Molecular Microbiology Activities Previously published probing primers (Horwood, Burgess & Oakey 2004) were synthesized at Integrated DNA Technologies, Coralville, IA, USA. They were employed against the genomic DNA of Arthrobacter nicotianae strain PR. The Mastercycler personal thermocycler (Eppendorf) was used to make the polymerase chain reaction (PCR) with a preparation of 2μL of DNA, 10mL of Premix Taq (catalog #RR003, Takara), 2μL of primer (forward and reverse), and 6uL of double distilled water in a 0.5mL microcentrifuge tube. The PCR conditions were based on the protocols that had been standardized earlier (Rajendran, 1999; Rajendran, Rajnarayanan, & Demuth, 2008). The following conditions: 95 oC (5 min), 95 oC (1 min), 55 oC (1 min), and 72 oC (3 min) for 30 cycles were adopted. The PCR amplicons were analyzed via 1% agarose gel electrophoresis using 100 bp DNA step ladder (catalog # G6951, Promega, Madison WI, USA). Before running electrophoresis, the gel was prepared by mixing 0.5g of Agarose with 50mL of TBE and 1μL of Ethidium bromide. The solution was then poured into the gel casting cassette (Thermo Scientific, USA). Loading dye (6μL) was added to each tube of the PCR products and loaded into the gel slots. The gel was run at 90V for about an hour. By using ‘Digidoc-it UVP digital documentation unit’, the profile of the amplicons were examined and documented. Individual PCR amplicon was gel extracted using PureLink Quick Gel Extraction kit (Catalog # K2100-12, Invitrogen, Carlsbad CA, USA). In brief, agarose gel with amplicon DNA was dissolved in gel solubilization solution (GS1) at 65C. The solution was passed through quick gel extraction column, which retained the DNA. Wash buffer (W9) was also added to the column before eluting with