A Call to Develop Course-Based Undergraduate Research Experiences (CUREs) for Nonmajors Courses.

Course-based undergraduate research experiences (CUREs) for non-science majors (nonmajors) are potentially distinct from CUREs for developing scientists in their goals, learning objectives, and assessment strategies. While national calls to improve science, technology, engineering, and mathematics education have led to an increase in research revealing the positive effects of CUREs for science majors, less work has specifically examined whether nonmajors are impacted in the same way. To address this gap in our understanding, a working group focused on nonmajors CUREs was convened to discuss the following questions: 1) What are our laboratory-learning goals for nonmajors? 2) What are our research priorities to determine best practices for nonmajors CUREs? 3) How can we collaborate to define and disseminate best practices for nonmajors in CUREs? We defined three broad student outcomes of prime importance to the nonmajors CURE: improvement of scientific literacy skills, proscience attitudes, and evidence-based decision making. We evaluated the state of knowledge of best practices for nonmajors, and identified research priorities for the future. The report that follows is a summary of the conclusions and future directions from our discussion.

Using Bioinformatics to Develop and Test Hypotheses: E. coli-Specific Virulence Determinants.

Bioinformatics, the use of computer resources to understand biological information, is an important tool in research, and can be easily integrated into the curriculum of undergraduate courses. Such an example is provided in this series of four activities that introduces students to the field of bioinformatics as they design PCR based tests for pathogenic E. coli strains. A variety of computer tools are used including BLAST searches at NCBI, bacterial genome searches at the Integrated Microbial Genomes (IMG) database, protein analysis at Pfam and literature research at PubMed. In the process, students also learn about virulence factors, enzyme function and horizontal gene transfer. Some or all of the four activities can be incorporated into microbiology or general biology courses taken by students at a variety of levels, ranging from high school through college. The activities build on one another as they teach and reinforce knowledge and skills, promote critical thinking, and provide for student collaboration and presentation. The computer-based activities can be done either in class or outside of class, thus are appropriate for inclusion in online or blended learning formats. Assessment data showed that students learned general microbiology concepts related to pathogenesis and enzyme function, gained skills in using tools of bioinformatics and molecular biology, and successfully developed and tested a scientific hypothesis.

Quantitative analysis of group II intron expression and splicing in Lactococcus lactis.

The group II intron Ll.ltrB is found within the ltrB relaxase gene of the conjugative element pRS01 in Lactococcus lactis. Precise splicing of the intron is essential for pRS01 transfer. The transcription regulation and in vivo splicing activity of Ll.ltrB have not been investigated thoroughly in L. lactis in the natural pRS01 context. We developed absolute quantitative real-time reverse transcription-PCR assays to quantify RNA levels of the 5' exon (ltrBE1) and the spliced relaxase (ltrB) and intron-encoded protein (ltrA) genes, as well as Ll.ltrB splicing activity under different physiological conditions. The mRNA levels for the ATP-binding protein OppD were assayed for comparison to the ltrB transcripts. The oppD mRNA ranged from 10- to 10,000-fold higher than ltrB region genes. ltrBE1 expression was growth-phase dependent. The mRNA level of ltrA was almost constant during all growth phases and in all media tested. Ll.ltrB in vivo splicing activity ranged from (6.5 +/- 2.1)% to (22.1 +/- 8.0)%. Acid challenge significantly decreased both ltrB region mRNA levels and intron splicing activity. The presence of recipient cells, different mating environments, and temperature stress had no significant effects on expression and splicing. Western blotting showed that the level of LtrB protein expressed from an intronless ltrB gene was much higher (about 20-fold) than the level of protein expressed from an intron-containing construct. Interestingly, LtrB protein showed a tendency to function in cis on its oriT target. The low level of ltrB transcript and relatively inefficient splicing of the intron may limit Ll.ltrB mobility and dissemination in nature.

Characterization of the pheromone response of the Enterococcus faecalis conjugative plasmid pCF10: complete sequence and comparative analysis of the transcriptional and phenotypic responses of pCF10-containing cells to pheromone induction.

The sex pheromone plasmids in Enterococcus faecalis are one of the most efficient conjugative plasmid transfer systems known in bacteria. Plasmid transfer rates can reach or exceed 10(-1) transconjugants per donor in vivo and under laboratory conditions. We report the completion of the DNA sequence of plasmid pCF10 and the analysis of the transcription profile of plasmid genes, relative to conjugative transfer ability following pheromone induction. These experiments employed a mini-microarray containing all 57 open reading frames of pCF10 and a set of selected chromosomal genes. A clear peak of transcription activity was observed 30 to 60 min after pheromone addition, with transcription subsiding 2 h after pheromone induction. The transcript activity correlated with the ability of donor cells to transfer pCF10 to recipient cells. Remarkably, aggregation substance (Asc10, encoded by the prgB gene) was present on the cell surface for a long period of time after pheromone-induced transcription of prgB and plasmid transfer ability had ceased. This observation could have relevance for the virulence of E. faecalis.

A conjugation-based system for genetic analysis of group II intron splicing in Lactococcus lactis.

The conjugative element pRS01 from Lactococcus lactis encodes the putative relaxase protein LtrB. The ltrB gene is interrupted by the functional group II intron Ll.ltrB. Accurate splicing of the two ltrB exons is required for synthesis of the mRNA encoding the LtrB conjugative relaxase and subsequent plasmid transfer. A conjugation-based genetic assay was developed to identify Ll.ltrB mutations that affect splicing. In this assay a nonsplicing, transfer-defective pRS01 derivative (pM1014) and a shuttle vector carrying the ltrB region, including the Ll.ltrB intron (pCOM9), are used. pCOM9 provides splicing-dependent complementation of the transfer defect of pM1014. Site-directed mutations within Ll.ltrB, either in the catalytic RNA or in the intron-encoded protein gene ltrA, were generated in the context of pCOM9. When these mutants were tested in the conjugation-based assay, significantly reduced mating was observed. Quantitative molecular analysis of in vivo splicing activity confirmed that the observed mating defects resulted from reduced splicing. Once the system was validated for the engineered mutants, random mutagenesis of the intron followed by genetic and molecular screening for splicing defects resulted in identification of point mutations that affect splicing.

Bacterial group II introns and their association with mobile genetic elements.

Group II introns are an abundant class of self-splicing RNAs, found primarily in the organelles of plants and lower eukaryotes and in bacteria. The first bacterial group II intron identified to be functional for splicing in vivo was the Ll.ltrB intron of Lactococcus lactis. It has served as an excellent model for the study of group II intron structure and function. Taking advantage of the tools of bacterial genetics and biochemical methodologies, details of Ll.ltrB splicing and homing reactions have been elucidated and are similar to those of fungal group II introns. This review provides a summary of these results. Of particular interest is the potential use of Ll.ltrB as an agent for targeted gene disruption. In addition, the development of a genetic system to analyze Ll.ltrB splicing promises to provide new insight into group II intron structure and function. Identification and analysis of group II introns in other bacterial species is a continuing process, and a discussion of published reports on these introns is provided here. Limited functional data is available for most of these introns, but sequence analysis points out several common themes, most notably that bacterial group II introns are almost always carried on mobile genetic elements.