Student Centered Education Remembering the Forest While Viewing the Trees: Evolutionary Thinking in the Teaching of Molecular Biology
Sitaraman Saraswati† Ramakrishnan Sitaraman‡*
From the †Department of Biochemistry, Dayananda Sagar Institutions, Shavige Malleshwara Hills, Kumaraswamy Layout, Bengaluru 560078, Karnataka, India, ‡Department of Biotechnology, TERI University, 10 Institutional Area, Vasant Kunj, New Delhi 110016, India
Abstract Given the centrality of evolutionary theory to the study of biology, we present a strategy for reinforcing its importance by appropriately recontextualizing classic and wellknown experiments that are not explicitly linked with evolution in conventional texts. This exercise gives students
an appreciation of the applicability of the theory of evolution in diverse contexts, including those where it is not C 2013 by The International Union of explicitly mentioned. V Biochemistry and Molecular Biology, 42(2):162–164, 2014.
Keywords: evolution; natural selection; molecular biology; teaching and learning techniques (methods and approaches)
Introduction Evolution by natural selection probably constitutes the one “law” that biology can uniquely lay claim to. Within the teaching of molecular biology, evolutionary theory is extensively used to interpret the results of homology searches, and facilitate the preparation of phylogenetic trees. However, in our admittedly limited and anecdotal experience, this often leads to an involuntary and unintended disconnect between the importance of evolutionary considerations and the biological systems under study in several other contexts. To address this problem, we have formulated a lesson plan in the form of questions and answers that encourages students to discover for themselves the evolutionary underpinnings of three classical experiments usually covered in introductory lectures/courses. However, it must be borne in mind that the overall plan given here assumes that evolution has been covered as a separate topic before commencing the following modules. Integrating diverse aspects of evolutionary theory and interpretation into the teaching of classical and well-known experiments that are included in, and indispensable to, life
*Address for corresponding to: Department of Biotechnology, TERI University, 10 Institutional Area, Vasant Kunj, New Delhi 110070, India. E-mail: [email protected] Received 9 September 2013; Accepted 30 October 2013 DOI 10.1002/bmb.20763 Published online 20 December 2013 in Wiley Online Library (wileyonlinelibrary.com)
162
sciences curricula applicability.
worldwide,
should
have
wide
Module 1: Bacterial Transformation Frederick Griffith’s classic experiments with “rough” (R, avirulent) and “smooth” (S, virulent) capsular types of Streptococcus pneumoniae [1] are too well-known to require a full description here. After going through the details, we ask our students the following questions and require them to restate and explore their answers in evolutionary terms: Q1. Why are no R-type cells recovered after coinfection of mice with live R-type and heat-killed S-type bacteria? A1. The usual answer is that the avirulent type (R) is cleared by the host immune system. Only those R-type bacteria that have been transformed to the S-type proliferate in the host. Q2. Restate your answer in evolutionary terms. What does this tell you about the “environment” that supplies the necessary selection pressure? A2. Here, we encourage our students to reframe the terms of the discourse as follows. For the infecting bacteria, the host immune response constitutes a selective agent that discriminates against R-type cells. Therefore, only transformants (S-type) survive passage through the host. The selection pressure can originate from not only abiotic, but also biotic agents (the immune system in this case). Q3. Is there any other characteristic that has been selected for during this experiment?
Biochemistry and Molecular Biology Education
A3. The selection process, that is, passage through a mammalian host, has not only enriched for bacteria having enhanced resistance to immune clearance by the host but incidentally also for those that possess natural competence enabling the uptake of exogenous DNA, that is, are capable of being transformed.
Module 2: Bacterial Conjugation Lederberg and Tatum’s landmark experiments with double auxotrophic mutants of E. coli K-12 provided the first indication of a mechanism of genetic transfer different from transformation or transduction (now known to be conjugation) occurring in bacteria [2]. They grew mixed cultures of double auxotrophs of E. coli K-12, one genotypcially met–bio–thr1pro1 and the other met1bio1thr–pro–, in liquid minimal medium supplemented with amino acids and vitamins. Aliquots of these mixed cultures were then plated on minimal medium, but without supplementation. The appearance of colonies on minimal medium indicated that some kind of genetic transfer had taken place, resulting in the appearance of recombinant, prototrophic bacteria. Q1. In evolutionary terms, are mutations resulting in auxotrophy necessarily deleterious? A1. In supplemented media, neither auxotroph has a competitive advantage, indicating the important fact that mutations, even somewhat potentially disadvantageous ones that result in auxotrophy, may persist in populations in the absence of appropriate selective pressures that weed them out. Q2. Depending on one’s viewpoint, what types of selection are possible? A2. One could have both positive and negative selection. On minimal medium without supplements, the auxotrophs are subjected to negative selection and prototrophs (transconjugants) to positive selection. Q3. Are there any biological characteristics (phenotypes) that are being positively selected in this experiment, in addition to prototrophy? A3. Yes, selection on minimal media without supplementation incidentally enriches for bacteria that possess the machinery for conjugation and genetic recombination that, in turn, allows for conjugation and the development of prototrophy respectively.
Module 3: The Three-Letter Genetic Code In 1961, Crick et al. published a remarkable paper [3] that concluded that the genetic code had to consist of three letters (or a multiple thereof), using purely genetic methods based on bacteriophage T4. Using an array of T4 mutants and simple phenotypic screens (plaque morphology and
growth on both B and K-12(k1) strains of E. coli or on the B strain alone observed with wild type and rII mutants, respectively), they were able to isolate mutant phages that had single point mutations as well as double mutants that were inferred to have suppressors of the initial point mutations. Analysis of successive suppressor mutations of the same type (single base insertions or deletions leading up to triple mutants) indicated at least three base additions or deletions were required to restore the reading frame, resulting in a “pseudo wild-type” phenotype that seemed to mimic the wild-type very closely. Q1. In the context of T4 rII mutants, does evolution necessarily improve or optimize the biological system? A1. As long as mutant phages can infect at least one strain of bacterial host, mutants will survive and proliferate just as well as the wild type. Although mutants are always arising in the population, a fortuitous circumstance (the availability of a suitable host) is all that is required for a particular mutant to propagate itself. If surrounded by E. coli B, the mutant is in no way impaired in its ability to infect new hosts and survive. Its presence may be inferred only when it is presented with the K-12(k1) strain as the sole available host. Therefore, “optimization” or “improvement” is never an a priori evolutionary consideration. It is the interaction of mutants with the selection pressures of the moment (e.g., presence of the K-12(k1) strain rather than B) that determine alternative outcomes. Q2. What can the phage T4 system tell us about the range of observable mutations for a particular gene? A2. The observation of mutations in any experimental system is heavily dependent on the nature of the locus that the mutations occur in—whether or not it is an essential gene and the extent to which mutated portions compromise critical biological functions. In that sense, the choice of the rII region was most fortuitous because mutations in this region gave clearly different plaque morphologies and differential ability to grow on bacterial host strains, but without compromising essential phage functions that ensured fitness, that is, the ability to produce offspring, in at least one host strain.
Discussions We have used the foregoing exercises to illustrate to our students not only the centrality of evolutionary considerations in biology, but also the more practical aspect of interpreting research papers from multiple perspectives, which stimulates creative lines of inquiry. Although the authors of research papers are correct to highlight particular aspects that they choose in the interests of coherence, relevance, and even brevity (journal articles have strict word limits), there are a number of unstated, implicit assumptions and prior knowledge [4] that lend credibility to their hypotheses and experimental methods, facilitating the development of ideas. Here, we have indicated how the major, yet often
163
Biochemistry and Molecular Biology Education implicit, theme of evolution may be brought into sharp relief in the analysis of three classic experiments in molecular biology that present very different explicit outcomes in widely varying contexts. As stated earlier, the topics were chosen because they form the staple of practically every academic degree program in the life sciences today. Even more to the point, most students are already familiar with the material which, in our experience, translates to more receptivity and less diffidence among them than would be the case if an entirely new topic were to be presented. The questions and answers listed in the above modules serve as useful outlines for stimulating class discussion and questioning. Therefore, these themes may be profitably used to highlight important aspects of evolutionary interpretation, and also enhance class participation in a dialogic process. An additional point is that such exercises also have the merit of presenting biology (molecular or otherwise) as a deductive, and not solely as a descriptive science. Our experience indicates that such an approach enables the seamless integration of evolutionary thinking into existing curricula without undue perturbation. The incorporation of evolutionary perspectives into our treatment of these
164
classic experiments creates renewed appreciation for Theodosius Dobzhansky’s famous declaration that “Nothing makes sense in biology except in the light of evolution” [5].
Acknowledgements This paper is dedicated to the authors’ parents, Mr. G. Sitaraman and Ms. Indubala, for active encouragement and support of their academic endeavours. The authors thank two anonymous reviewers for their insightful comments and suggestions. The authors have no conflicts of interest to declare.
References [1] Griffith, F. (1928) The significance of pneumococcal types. J. Hyg. (Lond.) 27, 113–159. [2] Tatum, E. L. and Lederberg, J. (1947) Gene recombination in the bacterium Escherichia coli. J. Bacteriol. 53, 673–684. [3] Crick, F. H. C., Barnett, L., Brenner, S., and Watts-Tobin, R. J. (1961) General nature of the genetic code for proteins. Nature 192, 1227–1232. [4] Rangachari, P. K. (2008) Of tacit knowledge, texts and thing-based learning (TBL). Biochem. Mol. Biol. Educ. 36, 363–364. [5] Dobzhansky, T. (1973) Nothing in biology makes sense except in the light of evolution. Am. Biol. Teach. 35, 125–129.
Student Centered Education
Sitaraman Saraswati† Ramakrishnan Sitaraman‡*
From the †Department of Biochemistry, Dayananda Sagar Institutions, Shavige Malleshwara Hills, Kumaraswamy Layout, Bengaluru 560078, Karnataka, India, ‡Department of Biotechnology, TERI University, 10 Institutional Area, Vasant Kunj, New Delhi 110016, India
Abstract Given the centrality of evolutionary theory to the study of biology, we present a strategy for reinforcing its importance by appropriately recontextualizing classic and wellknown experiments that are not explicitly linked with evolution in conventional texts. This exercise gives students
an appreciation of the applicability of the theory of evolution in diverse contexts, including those where it is not C 2013 by The International Union of explicitly mentioned. V Biochemistry and Molecular Biology, 42(2):162–164, 2014.
Keywords: evolution; natural selection; molecular biology; teaching and learning techniques (methods and approaches)
Introduction Evolution by natural selection probably constitutes the one “law” that biology can uniquely lay claim to. Within the teaching of molecular biology, evolutionary theory is extensively used to interpret the results of homology searches, and facilitate the preparation of phylogenetic trees. However, in our admittedly limited and anecdotal experience, this often leads to an involuntary and unintended disconnect between the importance of evolutionary considerations and the biological systems under study in several other contexts. To address this problem, we have formulated a lesson plan in the form of questions and answers that encourages students to discover for themselves the evolutionary underpinnings of three classical experiments usually covered in introductory lectures/courses. However, it must be borne in mind that the overall plan given here assumes that evolution has been covered as a separate topic before commencing the following modules. Integrating diverse aspects of evolutionary theory and interpretation into the teaching of classical and well-known experiments that are included in, and indispensable to, life
*Address for corresponding to: Department of Biotechnology, TERI University, 10 Institutional Area, Vasant Kunj, New Delhi 110070, India. E-mail: [email protected] Received 9 September 2013; Accepted 30 October 2013 DOI 10.1002/bmb.20763 Published online 20 December 2013 in Wiley Online Library (wileyonlinelibrary.com)
162
sciences curricula applicability.
worldwide,
should
have
wide
Module 1: Bacterial Transformation Frederick Griffith’s classic experiments with “rough” (R, avirulent) and “smooth” (S, virulent) capsular types of Streptococcus pneumoniae [1] are too well-known to require a full description here. After going through the details, we ask our students the following questions and require them to restate and explore their answers in evolutionary terms: Q1. Why are no R-type cells recovered after coinfection of mice with live R-type and heat-killed S-type bacteria? A1. The usual answer is that the avirulent type (R) is cleared by the host immune system. Only those R-type bacteria that have been transformed to the S-type proliferate in the host. Q2. Restate your answer in evolutionary terms. What does this tell you about the “environment” that supplies the necessary selection pressure? A2. Here, we encourage our students to reframe the terms of the discourse as follows. For the infecting bacteria, the host immune response constitutes a selective agent that discriminates against R-type cells. Therefore, only transformants (S-type) survive passage through the host. The selection pressure can originate from not only abiotic, but also biotic agents (the immune system in this case). Q3. Is there any other characteristic that has been selected for during this experiment?
Biochemistry and Molecular Biology Education
A3. The selection process, that is, passage through a mammalian host, has not only enriched for bacteria having enhanced resistance to immune clearance by the host but incidentally also for those that possess natural competence enabling the uptake of exogenous DNA, that is, are capable of being transformed.
Module 2: Bacterial Conjugation Lederberg and Tatum’s landmark experiments with double auxotrophic mutants of E. coli K-12 provided the first indication of a mechanism of genetic transfer different from transformation or transduction (now known to be conjugation) occurring in bacteria [2]. They grew mixed cultures of double auxotrophs of E. coli K-12, one genotypcially met–bio–thr1pro1 and the other met1bio1thr–pro–, in liquid minimal medium supplemented with amino acids and vitamins. Aliquots of these mixed cultures were then plated on minimal medium, but without supplementation. The appearance of colonies on minimal medium indicated that some kind of genetic transfer had taken place, resulting in the appearance of recombinant, prototrophic bacteria. Q1. In evolutionary terms, are mutations resulting in auxotrophy necessarily deleterious? A1. In supplemented media, neither auxotroph has a competitive advantage, indicating the important fact that mutations, even somewhat potentially disadvantageous ones that result in auxotrophy, may persist in populations in the absence of appropriate selective pressures that weed them out. Q2. Depending on one’s viewpoint, what types of selection are possible? A2. One could have both positive and negative selection. On minimal medium without supplements, the auxotrophs are subjected to negative selection and prototrophs (transconjugants) to positive selection. Q3. Are there any biological characteristics (phenotypes) that are being positively selected in this experiment, in addition to prototrophy? A3. Yes, selection on minimal media without supplementation incidentally enriches for bacteria that possess the machinery for conjugation and genetic recombination that, in turn, allows for conjugation and the development of prototrophy respectively.
Module 3: The Three-Letter Genetic Code In 1961, Crick et al. published a remarkable paper [3] that concluded that the genetic code had to consist of three letters (or a multiple thereof), using purely genetic methods based on bacteriophage T4. Using an array of T4 mutants and simple phenotypic screens (plaque morphology and
growth on both B and K-12(k1) strains of E. coli or on the B strain alone observed with wild type and rII mutants, respectively), they were able to isolate mutant phages that had single point mutations as well as double mutants that were inferred to have suppressors of the initial point mutations. Analysis of successive suppressor mutations of the same type (single base insertions or deletions leading up to triple mutants) indicated at least three base additions or deletions were required to restore the reading frame, resulting in a “pseudo wild-type” phenotype that seemed to mimic the wild-type very closely. Q1. In the context of T4 rII mutants, does evolution necessarily improve or optimize the biological system? A1. As long as mutant phages can infect at least one strain of bacterial host, mutants will survive and proliferate just as well as the wild type. Although mutants are always arising in the population, a fortuitous circumstance (the availability of a suitable host) is all that is required for a particular mutant to propagate itself. If surrounded by E. coli B, the mutant is in no way impaired in its ability to infect new hosts and survive. Its presence may be inferred only when it is presented with the K-12(k1) strain as the sole available host. Therefore, “optimization” or “improvement” is never an a priori evolutionary consideration. It is the interaction of mutants with the selection pressures of the moment (e.g., presence of the K-12(k1) strain rather than B) that determine alternative outcomes. Q2. What can the phage T4 system tell us about the range of observable mutations for a particular gene? A2. The observation of mutations in any experimental system is heavily dependent on the nature of the locus that the mutations occur in—whether or not it is an essential gene and the extent to which mutated portions compromise critical biological functions. In that sense, the choice of the rII region was most fortuitous because mutations in this region gave clearly different plaque morphologies and differential ability to grow on bacterial host strains, but without compromising essential phage functions that ensured fitness, that is, the ability to produce offspring, in at least one host strain.
Discussions We have used the foregoing exercises to illustrate to our students not only the centrality of evolutionary considerations in biology, but also the more practical aspect of interpreting research papers from multiple perspectives, which stimulates creative lines of inquiry. Although the authors of research papers are correct to highlight particular aspects that they choose in the interests of coherence, relevance, and even brevity (journal articles have strict word limits), there are a number of unstated, implicit assumptions and prior knowledge [4] that lend credibility to their hypotheses and experimental methods, facilitating the development of ideas. Here, we have indicated how the major, yet often
163
Biochemistry and Molecular Biology Education implicit, theme of evolution may be brought into sharp relief in the analysis of three classic experiments in molecular biology that present very different explicit outcomes in widely varying contexts. As stated earlier, the topics were chosen because they form the staple of practically every academic degree program in the life sciences today. Even more to the point, most students are already familiar with the material which, in our experience, translates to more receptivity and less diffidence among them than would be the case if an entirely new topic were to be presented. The questions and answers listed in the above modules serve as useful outlines for stimulating class discussion and questioning. Therefore, these themes may be profitably used to highlight important aspects of evolutionary interpretation, and also enhance class participation in a dialogic process. An additional point is that such exercises also have the merit of presenting biology (molecular or otherwise) as a deductive, and not solely as a descriptive science. Our experience indicates that such an approach enables the seamless integration of evolutionary thinking into existing curricula without undue perturbation. The incorporation of evolutionary perspectives into our treatment of these
164
classic experiments creates renewed appreciation for Theodosius Dobzhansky’s famous declaration that “Nothing makes sense in biology except in the light of evolution” [5].
Acknowledgements This paper is dedicated to the authors’ parents, Mr. G. Sitaraman and Ms. Indubala, for active encouragement and support of their academic endeavours. The authors thank two anonymous reviewers for their insightful comments and suggestions. The authors have no conflicts of interest to declare.
References [1] Griffith, F. (1928) The significance of pneumococcal types. J. Hyg. (Lond.) 27, 113–159. [2] Tatum, E. L. and Lederberg, J. (1947) Gene recombination in the bacterium Escherichia coli. J. Bacteriol. 53, 673–684. [3] Crick, F. H. C., Barnett, L., Brenner, S., and Watts-Tobin, R. J. (1961) General nature of the genetic code for proteins. Nature 192, 1227–1232. [4] Rangachari, P. K. (2008) Of tacit knowledge, texts and thing-based learning (TBL). Biochem. Mol. Biol. Educ. 36, 363–364. [5] Dobzhansky, T. (1973) Nothing in biology makes sense except in the light of evolution. Am. Biol. Teach. 35, 125–129.
Student Centered Education
