Using the two-hybrid screen in the classroom laboratory

The National Science Foundation and others have made compelling arguments that research be incorporated into the learning of undergraduates. In response to these arguments, a two-hybrid research project was incorporated into a molecular biology course that contained both a lecture section and a laboratory section. The course was designed around specific goals for educational outcomes, including introducing research to a wide range of students, teaching students experimental design and data analysis, and enhancing understanding of course material. Additional goals included teaching students to search genomic databases, to access scientific articles, and to write a paper in scientific format. Graded events tested these goals, and a student evaluation indicated student perception of the project. According to our analysis of the data, the yeast two-hybrid screen was a success: several novel clones were identified; students met expectations on graded lab reports, the poster session, and the final paper; and evaluations indicated that students had achieved the outlined goals. Students indicated on the evaluations that the research project increased their interest in research and greatly improved understanding of the course material. Finally, several students in the course intend to submit the findings of the research project to an undergraduate research journal.     

Comparing programmed cell death in cultured primary and tumor cells in inquiry-based cell biology laboratory exercises

ABSTRACT

Programmed cell death, or apoptosis, is a cellular pathway by which individual cells self-destruct for the benefit of the organism. In this practical paper, we describe laboratory exercises with an inquiry-based learning (IBL) approach in which undergraduate students compared apoptosis among different types of cultured cells. Ultraviolet (UV) radiation was used to induce apoptosis in mouse primary cells and in Chinese hamster ovary (CHO) tumor cells. Students hypothesized that, because tumor cells evade apoptosis, CHO cells would exhibit less apoptosis compared to primary cells. Treated and control cells were labeled for two hallmarks of apoptosis, fragmented DNA and active caspase-3 enzyme, and all nuclei were visualized with DAPI. Cell counts were conducted using fluorescence microscopy. Exposure to UV light induced apoptosis in both cell types compared to controls, but no significant difference in the proportions of labeled cells was observed between UV-treated primary and CHO cells. Optimal parameters for UV exposure and labeling techniques are presented. This exercise provides instructors with methodology for allowing students to use a basic cell culture system and microscopy to formulate a hypothesis and design experiments related to apoptosis. Students incorporated their work into a research paper, which served as the main mode of assessment.     

Multiweek cell culture project for use in upper-level biology laboratories

This article describes a laboratory protocol for a multiweek project piloted in a new upper-level biology laboratory (BIO 426) using cell culture techniques. Human embryonic kidney-293 cells were used, and several culture media and supplements were identified for students to design their own experiments. Treatments included amino acids, EGF, caffeine, epinephrine, heavy metals, and FBS. Students researched primary literature to determine their experimental variables, made their own solutions, and treated their cells over a period of 2 wk. Before this, a sterile technique laboratory was developed to teach students how to work with the cells and minimize contamination. Students designed their experiments, mixed their solutions, seeded their cells, and treated them with their control and experimental media. Students had the choice of manipulating a number of variables, including incubation times, exposure to treatment media, and temperature. At the end of the experiment, students observed the effects of their treatment, harvested and dyed their cells, counted relative cell numbers in control and treatment flasks, and determined the ratio of living to dead cells using a hemocytometer. At the conclusion of the experiment, students presented their findings in a poster presentation. This laboratory can be expanded or adapted to include additional cell lines and treatments. The ability to design and implement their own experiments has been shown to increase student engagement in the biology-related laboratory activities as well as develop the critical thinking skills needed for independent research.     

Laboratory demonstration of vascular smooth muscle function using rat aortic ring segments

This laboratory exercise uses a simple preparation and a straightforward protocol to illustrate many of the basic principles of vascular biology covered in an introductory physiology course. The design of this laboratory allows students to actively participate in an exercise demonstrating the regulation of arterial tone by endothelial and extrinsic factors. In addition, this hands-on laboratory allows students to gather data using well-known basic biomedical research techniques. Specifically, students are introduced to an isolated organ-chamber technique that is widely used to study cellular mechanisms of many tissues including vascular smooth muscle contraction and dilation. On the basis of student evaluations, participation in the experiments and interpreting data reinforce lecture materials on smooth muscle and endothelial cell function and illustrate mechanisms regulating vascular tone. Students come away with a greater understanding of vascular biology, a deeper appreciation of integrative physiology, and an understanding of the process of conducting tissue-chamber experiments.     

An approach to the critical assessment of the experimental conditions in practical molecular biology:: isolation of plant DNA

A plant molecular biology laboratory class experiment, based on real lab situations, is proposed to illustrate to the students how the assessment of the experimental conditions and the correct application of the technical details are critical also with an established procedure. During plant genomic DNA isolation and purification, a number of undesired effects, including DNA shearing and formation of secondary compounds, may take place. To simulate such events, we have set up a simple practical laboratory experiment in which the students are asked to isolate plant DNA under three different working conditions, from an optimal to a coarse one. The subsequent analyses on the extracted DNA enable the students to evaluate the influence of these different manipulations on DNA yield and quality. The experiment described can help the students to adopt a critical approach when interpreting the results of common preparative molecular techniques.     

Sex determination using the polymerase chain reaction

A practical class experiment on the PCR is described which has been used over several years as part of an undergraduate biochemistry and molecular biology course for science students. A major aim is to provide experience in the use of the polymerase chain reaction (PCR) and its interpretation. Students are given small coded DNA samples and use the PCR reaction to determine whether the sample is from a male or a female.     

Model of the developing tumorigenic phenotype in mammalian cells and the roles of sustained stress and replicative senescence

The molecular mechanisms that drive mammalian cells to the development of cancer are the subject of intense biochemical, genetic and medical studies. But for the present, there is no comprehensive model that might serve as a general framework for the interpretation of experimental data. This paper is an attempt to create a conceptual model of the mechanism of the developing tumorigenic phenotype in mammalian cells, defined as having high genomic instability and proliferative activity. The basic statement in the model is that mutations acquired by tumor cells are not caused directly by external DNA damaging agents, but instead are produced by the cell itself as an output of a Mutator Response similar to the bacterial "SOS response" and characterized by the initiation of error-prone cell cycle progression and an elevated rate of mutation. This response may be induced in arrested mammalian cells by intracellular and extracellular proliferative signals combined with blocked apoptosis. The mutant cells originated by this response are subjected to natural selection via apoptosis and turnover. This selection process favors the survival of cells with high proliferative activity and the suppression of apoptosis resulting in the long run in the appearance of immortalized cells with high proliferative activity. Either a sustained stressful environment accompanied by continuing apoptotic cell death, or replicative senescence, provides conditions suitable for activation of the Mutator Response, namely the emergence of arrested cells with blocked apoptosis and the induction of proliferative signal. It also accelerates the selection process by providing continuing cell turnover. The proposed mechanism is described at the level of involved metabolic pathways and proteins and substantiated by the related experimental data available in the literature.     

A linked series of laboratory exercises in molecular biology utilizing bioinformatics and GFP

Molecular biologists commonly use bioinformatics to map and analyze DNA and protein sequences and to align different DNA and protein sequences for comparison. Additionally, biologists can create and view 3D models of protein structures to further understand intramolecular interactions. The primary goal of this 10-week laboratory was to introduce the importance of bioinformatics in molecular biology. Students employed multiprimer, site-directed mutagenesis to create variant colors from a plasmid expressing green fluorescent protein (GFP). Isolated mutant plasmid from Escherichia coli showing changes in fluorescence were sequenced. Students used sequence alignment tools, protein translator tools, protein modeling, and visualization to analyze the potential effect of their mutations within the protein structure. This laboratory linked molecular techniques and bioinformatics to promote and expand the understanding of experimental results in an upper-level undergraduate laboratory course.     

Using C. elegans notch mutants in an undergraduate cancer biology laboratory course to demonstrate oncogenic effects on cell proliferation

Undergraduate laboratory exercises addressing aspects of cancer biology such as increased cell proliferation, gain-of-function signaling mutations and tumour formation often rely on tissue culture or even small mammal models. Many departments have limited or no access to these tools, and even well-equipped departments face logistical problems when incorporating these models into laboratory classes. I have developed a laboratory exercise using the microscopic worm, C. elegans, to demonstrate the effects of Notch receptor mutations on cell proliferation. Notch, which is activated by juxtacrine signaling, is mutated in many human cancers. In this exercise, students compare the germline phenotypes of worms that have a loss-of-function Notch mutation (no cells in the germline) or a gain-of-function Notch mutation (over-proliferation resulting in a germline tumour). Students also genotype the worms and perform sequence analysis to determine the effects of the mutations on the protein sequence. This laboratory exercise demonstrates oncogenic proliferation, correlates genotype to phenotype, exposes students to model organisms and introduces sequence databases and analysis. In addition to cancer biology courses, this exercise could be incorporated in courses with a focus on genetics, cell biology or developmental biology.     

An at‐home laboratory in plant biology designed to engage students in the process of science

The COVID‐19 pandemic prompted a transition to remote delivery of courses that lack immersive hands‐on research experiences for undergraduate science students, resulting in a scientific research skills gap. In this report, we present an option for an inclusive and authentic, hands‐on research experience that all students can perform off‐campus. Biology students in a semester‐long (13 weeks) sophomore plant physiology course participated in an at‐home laboratory designed to study the impacts of nitrogen addition on growth rates and root nodulation by wild nitrogen‐fixing Rhizobia in Pisum sativum (Pea) plants. This undergraduate research experience, piloted in the fall semester of 2020 in a class with 90 students, was created to help participants learn and practice scientific research skills during the COVID‐19 pandemic. Specifically, the learning outcomes associated with this at‐home research experience were: (1) generate a testable hypothesis, (2) design an experiment to test the hypothesis, (3) explain the importance of biological replication, (4) perform meaningful statistical analyses using R, and (5) compose a research paper to effectively communicate findings to a general biology audience. Students were provided with an at‐home laboratory kit containing the required materials and reagents, which were chosen to be accessible and affordable in case students were unable to access our laboratory kit. Students were guided through all aspects of research, including hypothesis generation, data collection, and data analysis, with video tutorials and live virtual sessions. This at‐home laboratory provided students an opportunity to practice hands‐on research with the flexibility to collect and analyze their own data in a remote setting during the COVID‐19 pandemic. This, or similar laboratories, could also be used as part of distance learning biology courses.     

人类血型性状综合遗传大实验的设计与教学实践

综合大实验能够训练学生灵活应用理论知识并掌握实验技能,成为当前实验课教学改革的重要方式。本文以人类的ABO血型性状为实验对象,设计了“人类ABO血型分子基因分型与群体遗传平衡分析”大实验。实验中提取同学唾液中黏膜细胞的DNA,经过PCR扩增目的片段、酶切及电泳分离一系列分子遗传技术分析,鉴定出每位同学的基因型;然后以全班同学为一个类似孟德尔群体调查ABO血型的各种基因型频数,用Popgene软件分析各种群体遗传参数。通过开放教学不仅让学生掌握了分子遗传实验技术和群体遗传分析技术及软件应用,还让学生自主设计方案优化分子技术环节,提高学生驾驭知识的能力。通过5年的教学探索与实践,建立了稳定的分子遗传实验体系,能够清楚地检测出ABO血型的6种基因型：I^AI^A、I^Ai、I^BI^B、I^Bi、I^AI^B、ii;综合了分子遗传与群体遗传的实验教学,统计全班同学6种基因型的频数,直接计算3个复等位基因的频率,进而应用软件分析群体遗传各种参数;实现了学生自主设计并完成实验的开放式实验教学;大实验教学获得了学生的好评,取得了很好的教学效果。该大实验可直接应用于生物类专业的遗传学实验教学,其中的教学理念和方法还可推广应用于其他生物学实验教学。     

Cytomics - importance of multimodal analysis of cell function and proliferation in oncology

Cancer is a highly complex and heterogeneous disease involving a succession of genetic changes (frequently caused or accompanied by exogenous trauma), and resulting in a molecular phenotype that in turn results in a malignant specification. The development of malignancy has been described as a multistep process involving self-sufficiency in growth signals, insensitivity to antigrowth signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and finally tissue invasion and metastasis. The quantitative analysis of networking molecules within the cells might be applied to understand native-state tissue signalling biology, complex drug actions and dysfunctional signalling in transformed cells, that is, in cancer cells. High-content and high-throughput single-cell analysis can lead to systems biology and cytomics. The application of cytomics in cancer research and diagnostics is very broad, ranging from the better understanding of the tumour cell biology to the identification of residual tumour cells after treatment, to drug discovery. The ultimate goal is to pinpoint in detail these processes on the molecular, cellular and tissue level. A comprehensive knowledge of these will require tissue analysis, which is multiplex and functional; thus, vast amounts of data are being collected from current genomic and proteomic platforms for integration and interpretation as well as for new varieties of updated cytomics technology. This overview will briefly highlight the most important aspects of this continuously developing field.     

Glucose transport in cultured animal cells: an exercise for the undergraduate cell biology laboratory

Membrane transport is a fundamental concept that undergraduate students of cell biology understand better with laboratory experience. Formal teaching exercises commonly used to illustrate this concept are unbiological, qualitative, or intricate and time consuming to prepare. We have developed an exercise that uses uptake of radiolabeled nutrient analogues by attachment-dependent animal cells cultured on multiwell trays. This system can readily be manipulated within a typical 3-h laboratory period to yield reproducible, biologically relevant, quantitative data regarding key aspects of membrane transport. Each 24-well tray of cultures allows a group of two to four students to compare eight conditions in triplicate. If different groups of students test different conditions or different types of cells, data can be shared for an even broader experience. The exercise is also readily adaptable for open-ended student projects. Here we illustrate the exercise measuring uptake of the nonmetabolizable glucose analogue [(3)H]-2-deoxy-D-glucose. Students successfully tested the effects of competing sugars, putative inhibitors of the GLUT1 transporter, and changes in cell physiology that might be expected to affect glucose transport in epithelial cells and fibroblasts. In this exercise students find the nutritional and medical implications of glucose transport and its regulation intriguing. They also learn to handle radioisotopes and cultured cells.     

Four decades of teaching developmental biology in Germany

I have taught developmental biology in Essen for 30 years. Since my department is named Zoophysiologie (Zoophysiology), besides Developmental Biology, I also have to teach General Animal Physiology. This explains why the time for teaching developmental biology is restricted to a lecture course, a laboratory course and several seminar courses. However, I also try to demonstrate in the lecture courses on General Physiology the close relationship between developmental biology, physiology, morphology, anatomy, teratology, carcinogenesis, evolution and ecology (importance of environmental factors on embryogenesis). Students are informed that developmental biology is a core discipline of biology. In the last decade, knowledge about molecular mechanisms in different organisms has exponentially increased. The students are trained to understand the close relationship between conserved gene structure, gene function and signaling pathways, in addition to or as an extension of, classical concepts. Public reports about the human genome project and stem cell research (especially therapeutic and reproductive cloning) have shown that developmental biology, both in traditional view and at the molecular level, is essential for the understanding of these complex topics and for serious and non-emotional debate.     

荧光显微成像在医学细胞生物学实验教学中的应用探索

医学细胞生物学是医学院校本科生的重要基础学科之一,其实验课程教学工作的改革与创新具有重要意义。本文深入分析了医学细胞生物学实验课程现状,认为课程普遍存在目的不明确、内容不新颖、缺乏整体性、临床联系少等问题。荧光显微成像技术是细胞生物学研究中的关键技术,利用这一技术设计具有整体性的科研实验对于培养医学生操作能力和科研素养具有重要意义。顺铂(cisplatin)为当前肿瘤联合化疗中最常用的药物之一,由于其抗癌机制涉及细胞增殖、细胞凋亡、细胞骨架等一系列细胞生物学知识,且与临床联系紧密,非常适合用于综合性实验设计。实验方案拟使用BrdU染色、Hoechst33258染色及鬼笔环肽染色法,分别检测人肺癌细胞系A549顺铂处理后细胞增殖、细胞凋亡及细胞骨架重排情况。实验完成后,鼓励学生通过查阅文献得出综合的实验结论。希望能够通过科研与教学相融合的方式,更好的激发医学生的学习热情,提高医学生的科研素质,培养全方位的医学人才。     

The Russian Biological Student Microscopes

Our out-of-school practical exercise was designed to bring upper secondary school students in contact with one of the most exciting and expanding topics in biology today: epigenetics. In school, students only study the basics in genetics and the respective investigation techniques as provided by the syllabus. For a practical exercise in epigenetics, however, they need additional knowledge. Hence, they are introduced to the subject of epigenetics and its molecular mechanisms. Students are asked to examine the different DNA methylation conditions of lambda DNA using both a restriction assay and gel electrophoresis in an out-of-school laboratory. DNA methylation is one of the major epigenetic mechanisms which have significant effects on gene expression; studies on monozygotic twins have shown that it is influenced by the environment. This exercise enables students to correctly identify the different methylation conditions of distributed lambda DNA samples. In doing so, they receive a first introduction to one epigenetic mechanism. The necessity for students to experience science in out-of-school settings has been shown by several scholars. The practical exercise we are proposing in this article was elaborated for such learning opportunities for upper secondary students to gain insight into contemporary science issues.     

Biotechnology apprenticeship for secondary-level students: teaching advanced cell culture techniques for research

The purpose of this article is to discuss small-group apprenticeships (SGAs) as a method to instruct cell culture techniques to high school participants. The study aimed to teach cell culture practices and to introduce advanced imaging techniques to solve various biomedical engineering problems. Participants designed and completed experiments using both flow cytometry and laser scanning cytometry during the 1-month summer apprenticeship. In addition to effectively and efficiently teaching cell biology laboratory techniques, this course design provided an opportunity for research training, career exploration, and mentoring. Students participated in active research projects, working with a skilled interdisciplinary team of researchers in a large research institution with access to state-of-the-art instrumentation. The instructors, composed of graduate students, laboratory managers, and principal investigators, worked well together to present a real and worthwhile research experience. The students enjoyed learning cell culture techniques while contributing to active research projects. The institution's researchers were equally enthusiastic to instruct and serve as mentors. In this article, we clarify and illuminate the value of small-group laboratory apprenticeships to the institution and the students by presenting the results and experiences of seven middle and high school participants and their instructors.     

Characterization of pathogenic human MSH2 missense mutations using yeast as a model system: a laboratory course in molecular biology

This work describes the project for an advanced undergraduate laboratory course in cell and molecular biology. One objective of the course is to teach students a variety of cellular and molecular techniques while conducting original research. A second objective is to provide instruction in science writing and data presentation by requiring comprehensive laboratory reports modeled on the primary literature. The project for the course focuses on a gene, MSH2, implicated in the most common form of inherited colorectal cancer. Msh2 is important for maintaining the fidelity of genetic material where it functions as an important component of the DNA mismatch repair machinery. The goal of the project has two parts. The first part is to create mapped missense mutation listed in the human databases in the cognate yeast MSH2 gene and to assay for defects in DNA mismatch repair. The second part of the course is directed towards understanding in what way are the variant proteins defective for mismatch repair. Protein levels are analyzed to determine if the missense alleles display decreased expression. Furthermore, the students establish whether the Msh2p variants are properly localized to the nucleus using indirect immunofluorescence and whether the altered proteins have lost their ability to interact with other subunits of the MMR complex by creating recombinant DNA molecules and employing the yeast 2-hybrid assay.