in this drawing of the lac operon, which molecule is an inactive repressor?

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Research Article | January 01 2015

Teaching the Big Ideas of Biology with Operon Models

The American Biology Teacher (2015) 77 (1): 30–39.

This paper presents an activity that engages students in model-based reasoning, requiring them to predict the behavior of the trp and lac operons under different environmental conditions. Students are presented six scenarios for the trp operon and five for the lac operon. In most of the scenarios, specific mutations have occurred in genetic elements of the system that alter the behavior from the norm. Students are also challenged to relate their understanding of operon behavior to the "Big Ideas" of homeostasis, evolution, information, interactions, and emergent properties. By using operons to teach students to reason with models of complex systems and understand broad themes, we equip them with powerful skills and ideas that form a solid foundation for their future learning in biology.

The essence of thinking and working scientifically involves modeling (Harrison & Treagust, 2000). For students, developing the ability to reason with scientific models is an integral part of scientific literacy (Gobert & Buckley, 2000; Gilbert, 2011). Here, I present an activity that requires students to reason with two operon models, challenging them to develop a deeper understanding of how operons function by predicting the behavior of the trp and lac operons under varying conditions. In addition, students are prompted to make connections to major themes of biology, the "Big Ideas" in the Advanced Placement (AP) Biology Framework (College Board, 2012). The same major themes are also found in the Next Generation Science Standards (NGSS Lead States, 2013) and in Vision and Change (AAAS, 2011). The activity is appropriate for advanced high school, AP Biology, and introductory undergraduate levels.

Models & Model-based Reasoning

A model is a "system of objects, symbols, and relationships representing another system (called a target) in a different medium" (Gilbert, 2011, p. 3). Models come in a variety of forms, but in each case, the model constructed represents only those elements of a system necessary to fulfill the purposes of the modeler. Constructing models is fundamental to the way both scientists and nonscientists think about the world (Collins & Gentner, 1987). As we interact with objects and people around us, we construct mental models that enable us to make sense of the world and, to a degree, predict what will happen in various situations. Models are based on analogies and, as such, they can mislead, but when the models fail, they also provide the foundation for learning through revision of the model (Collins & Gentner, 1987). Scientists are much more deliberate in their model construction than nonscientists, and scientific models are socially constructed to a greater degree than are the idiosyncratic, personal mental models that people construct and use on a daily basis. However, both personal mental models and scientific models serve similar functions: understanding and prediction.

The trp and lac operon models used in this activity (see Figures 1 and 2) are scientific models that support reasoning about the coordinated interactions of genes, proteins, and small molecules in the bacterium Escherichia coli. The lac operon model developed by François Jacob and Jacques Monod is historically important in that it provided the foundation for reasoning about the coordinated regulation of gene expression in many other organisms. But beyond the historical importance of the lac operon, operon models can serve an important pedagogical function. If students are to be scientifically literate, it is not sufficient for them to merely memorize and recite facts. Like scientists, they must learn to reason with models (Ohlsson, 1992; Gilbert, 2011). Developing students' abilities in model-based reasoning is a scientific practice that is advocated in all the recently released documents guiding the reform of science education (AAAS, 2011; College Board, 2012; NGSS Lead States, 2013).

Figure 1.

Figure 1. The trp operon.

The trp operon.

Figure 1.

Figure 1. The trp operon.

The trp operon.

Figure 2.

Figure 2. The lac operon.

The lac operon.

Figure 2.

Figure 2. The lac operon.

The lac operon.

The Activity

The activity is intended for use in a unit on gene regulation, and considerable prior knowledge is necessary for students to be successful. Students should be familiar with basic concepts of molecular genetics, including transcription and translation. They should be able to distinguish the various types of point mutations, including substitutions, insertions, and deletions, and their effects on protein structure and function. In addition, students should be familiar with the allosteric regulation of proteins. Finally, they should have had prior exposure to major biological themes common to the AP Biology Framework, the Next Generation Science Standards, and Vision and Change, including interactions, flow of matter and energy, emergent properties, homeostasis, evolution, and information storage and transfer.

The activity has three phases: (1) direct instruction on operon structure and function, (2) a problem-solving exercise to promote student discussion and deeper processing, and (3) reflection on how the major themes of biology make sense of the structure and function of operons. In order to make the best use of class time, much of the direct instruction can be accomplished with homework outside of class, freeing class time for student–student and student–instructor interaction. The value of this activity depends on the quality of interaction that occurs in phases 2 and 3. The scenarios posed in phase 2 create a problem space that promotes student reasoning, question generation, and discussion with both peers and the instructor. Ideally, the activity should engage students at a level that requires productive struggle to solve the problems within what Vygotsky (1978) called their "zone of proximal development." Sample problems are provided in Tables 1 and 2 that are appropriate for AP or undergraduate levels. However, by manipulating the variables in the systems, the level of difficulty of the problems can be adjusted to accommodate students at different ability levels.

Table 1.

The trp operon. Circle the terms in each box that correctly describe the state of the operon elements under the conditions.

Condition of Genes in the System	Environmental Conditions
	No Tryptophan Present			Tryptophan Present
A There are no mutations in the genes. All control mechanisms and structural genes are functioning normally.	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized
B There is a frameshift mutation resulting from a deletion of one base early in the sequence of the regulatory gene (trp R).	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized
C There is a substitution mutation in the operator preventing the binding of the repressor protein at the operator site.	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized
D There is a substitution resulting in a nonsense mutation early in the sequence of structural gene trp C which produces an enzyme in the pathway for tryptophan synthesis.	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized
E There is a silent mutation in the regulatory gene (trp R).	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized
F There is a missense substitution mutation in the regulatory gene (trp R) which produces a protein with slightly altered conformation. The resulting protein cannot bind the corepressor tryptophan.	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized

Condition of Genes in the System	Environmental Conditions
	No Tryptophan Present			Tryptophan Present
A There are no mutations in the genes. All control mechanisms and structural genes are functioning normally.	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized
B There is a frameshift mutation resulting from a deletion of one base early in the sequence of the regulatory gene (trp R).	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized
C There is a substitution mutation in the operator preventing the binding of the repressor protein at the operator site.	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized
D There is a substitution resulting in a nonsense mutation early in the sequence of structural gene trp C which produces an enzyme in the pathway for tryptophan synthesis.	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized
E There is a silent mutation in the regulatory gene (trp R).	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized
F There is a missense substitution mutation in the regulatory gene (trp R) which produces a protein with slightly altered conformation. The resulting protein cannot bind the corepressor tryptophan.	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized	Repressor: Operon is: Tryptophan	Active (=Binding to DNA) Off (=genes not transcribed) Not Synthesized	Inactive On Synthesized

Answers to Table 1: trp Operon

Condition A: No Tryptophan (inactive, on, synthesized), Tryptophan (active, off, not synthesized); Condition B: No Tryptophan (inactive, on, synthesized), Tryptophan (inactive, on, synthesized); Condition C: No Tryptophan (inactive, on, synthesized), Tryptophan (inactive, on, synthesized); Condition D: No Tryptophan (inactive, on, not synthesized), Tryptophan (active, off, not synthesized); Condition E: No Tryptophan (inactive, on, synthesized), Tryptophan (active, off, not synthesized); Condition F: No Tryptophan (inactive, on, synthesized), Tryptophan (inactive, on, synthesized).

Additional Notes on Table 1: In Condition C, when tryptophan is present the repressor will assume an active conformation, but will be unable to bind to the mutated operator; therefore, it is effectively inactive. In Condition D, trp C is a structural gene that produces an enzyme necessary for tryptophan synthesis. The operon will function normally, but no tryptophan will be synthesized because of the enzyme missing from the metabolic pathway.

Table 2:

The lac Operon. Circle the terms in each box that correctly describe state of the operon elements under the conditions.

Condition of Genes in the System	Glucose in Environment	Environmental Conditions
		No Lactose present			Lactose present
A There is a mutation in the regulatory gene (lac I) that results in a repressor protein that binds to the operator, but cannot bind the inducer allolactose.	No Glucose	cAMP level: CAP Activity: Repressor: Operon is:	High Active (=Binding to DNA) Active (=Binding to DNA) Off On low (genes	None Inactive Inactive On high transcribed)	cAMP level: CAP Activity: Repressor: Operon is:	High Active (=Binding to DNA) Active(=Binding to DNA) Off On low (genes	None Inactive Inactive On high transcribed)
B There is a nonsense mutation in the regulatory gene (lac I) resulting in a premature stop at the 30^th codon of the mRNA molecule that codes for the protein. The functional lac repressor has 4 identical subunits each of which has 360 amino acids.	No Glucose	cAMP level: CAP Activity: Repressor: Operon is:	High Active (=Binding to DNA) Active (=Binding to DNA) Off On low (genes	None Inactive Inactive On high transcribed)	cAMP level: CAP Activity: Repressor: Operon is:	High Active (=Binding to DNA) Active (=Binding to DNA) Off On low (genes	None Inactive Inactive On high transcribed)
C There is a single base insertion early in the sequence of the lac Z gene that codes for the enzyme β-galactosidase.	No Glucose	cAMP level: CAP Activity: Repressor: Operon is:	High Active (=Binding to DNA) Active (=Binding to DNA) Off On low (genes	None Inactive Inactive On high transcribed)	cAMP level: CAP Activity: Repressor: Operon is:	High Active (=Binding to DNA) Active (=Binding to DNA) Off On low (genes	None Inactive Inactive On high transcribed)
D There is a mutation in the operator preventing the recognition and binding of the lac repressor regulatory protein at the operator site.	High Glucose Level	cAMP level: CAP Activity: Repressor: Operon is:	High Active (=Binding to DNA) Active (=Binding to DNA) Off On low (genes	None Inactive Inactive On high transcribed)	cAMP level: CAP Activity: Repressor: Operon is:	High Active (=Binding to DNA) Active (=Binding to DNA) Off On low (genes	None Inactive Inactive On high transcribed)
E There is a nonsense mutation early in the sequence of the lac Y gene.	No Glucose	cAMP level: CAP Activity: Repressor: Operon is:	High Active (=Binding to DNA) Active (=Binding to DNA) Off On low (genes	None Inactive Inactive On high transcribed)	cAMP level: CAP Activity: Repressor: Operon is:	High Active (=Binding to DNA) Active (=Binding to DNA) Off On low (genes	None Inactive Inactive On high transcribed)

Condition of Genes in the System	Glucose in Environment	Environmental Conditions
		No Lactose present			Lactose present
A There is a mutation in the regulatory gene (lac I) that results in a repressor protein that binds to the operator, but cannot bind the inducer allolactose.	No Glucose	cAMP level: CAP Activity: Repressor: Operon is:	High Active (=Binding to DNA) Active (=Binding to DNA) Off On low (genes	None Inactive Inactive On high transcribed)	cAMP level: CAP Activity: Repressor: Operon is:	High Active (=Binding to DNA) Active(=Binding to DNA) Off On low (genes	None Inactive Inactive On high transcribed)
B There is a nonsense mutation in the regulatory gene (lac I) resulting in a premature stop at the 30^th codon of the mRNA molecule that codes for the protein. The functional lac repressor has 4 identical subunits each of which has 360 amino acids.	No Glucose	cAMP level: CAP Activity: Repressor: Operon is:	High Active (=Binding to DNA) Active (=Binding to DNA) Off On low (genes	None Inactive Inactive On high transcribed)	cAMP level: CAP Activity: Repressor: Operon is:	High Active (=Binding to DNA) Active (=Binding to DNA) Off On low (genes	None Inactive Inactive On high transcribed)
C There is a single base insertion early in the sequence of the lac Z gene that codes for the enzyme β-galactosidase.	No Glucose	cAMP level: CAP Activity: Repressor: Operon is:	High Active (=Binding to DNA) Active (=Binding to DNA) Off On low (genes	None Inactive Inactive On high transcribed)	cAMP level: CAP Activity: Repressor: Operon is:	High Active (=Binding to DNA) Active (=Binding to DNA) Off On low (genes	None Inactive Inactive On high transcribed)
D There is a mutation in the operator preventing the recognition and binding of the lac repressor regulatory protein at the operator site.	High Glucose Level	cAMP level: CAP Activity: Repressor: Operon is:	High Active (=Binding to DNA) Active (=Binding to DNA) Off On low (genes	None Inactive Inactive On high transcribed)	cAMP level: CAP Activity: Repressor: Operon is:	High Active (=Binding to DNA) Active (=Binding to DNA) Off On low (genes	None Inactive Inactive On high transcribed)
E There is a nonsense mutation early in the sequence of the lac Y gene.	No Glucose	cAMP level: CAP Activity: Repressor: Operon is:	High Active (=Binding to DNA) Active (=Binding to DNA) Off On low (genes	None Inactive Inactive On high transcribed)	cAMP level: CAP Activity: Repressor: Operon is:	High Active (=Binding to DNA) Active (=Binding to DNA) Off On low (genes	None Inactive Inactive On high transcribed)

Answers to Table 2: lac Operon

Condition A: No Lactose (cAMP High, CAP Active, Repressor Active, Operon Off), Lactose (cAMP High, CAP Active, Repressor Active, Operon Off); Condition B: No Lactose (cAMP High, CAP Active, Repressor inactive, Operon On high), Lactose (cAMP High, CAP Active, Repressor Inactive, Operon On high); Condition C: No Lactose (cAMP High, CAP Active, Repressor Active, Operon Off), Lactose (cAMP High, CAP Active, Repressor inactive, Operon On high); Condition D: No Lactose (cAMP None, CAP Inactive, Repressor inactive, Operon On low), Lactose (cAMP None, CAP Inactive, Repressor inactive, Operon On low); Condition E: No Lactose (cAMP High, CAP Active, Repressor Active, Operon Off), Lactose (cAMP High, CAP Active, Repressor Active, Operon Off).

Additional Notes: In Condition C, the operon functions normally since all regulatory elements are functional, but lactose will not be hydrolyzed when present since the gene for β-galactosidase produces a defective product. In Condition D, when there is no lactose, the operon will be on even though the repressor protein assumes an active conformation. The repressor is unable to bind to the operator and block transcription. Since glucose levels are high, the lac operon will only be on at a low level. In Condition E, the mutation in lac Y, the permease, will prevent transport of lactose across the plasma membrane, so although it may be abundant in the environment, it will not enter the cell, the operon will remain off and no lactose will be hydrolyzed.

As students work together to solve the problems, they construct personal mental models that support their reasoning about how operons work. Through discussion and defense of their reasoning with peers and the instructor, students develop a deeper understanding of operons that goes well beyond mere memorization of definitions. While students work in groups, the instructor has opportunities to answer student questions as they arise, listen in on student discussions to diagnose misconceptions, ask probing questions to check for understanding, and provide scaffolding necessary to promote student success. These interactions continue in phase 3 as students relate operon structure and function to the Big Ideas.

In the first phase, conducted outside of class, students read an excerpt from Carl Zimmer's Microcosm (2008, pp. 32–35) and a one-page editorial from the journal Science in which François Jacob (2011) reflects on his and Jacques Monod's discovery of the lac operon and its impact 50 years on. The readings serve to generate students' interest. In the passage from Microcosm, Zimmer engagingly describes how Jacob began research on the regulation of gene activity in E. coli that led ultimately to discovery of the lac operon. He characterizes Jacob's initial probing question as "Why genes sometimes make proteins and sometimes don't" (Zimmer, 2008, p. 32). The anecdotal information on Jacob's personal background and how he came to study the lac operon humanizes the scientist and provides historical context. This may interest high school and college students who are wrestling with decisions about their own careers and life goals. In addition, the anecdote shows how science can begin with the simplest of questions and ultimately lead to groundbreaking research.

After reading the excerpt, students should be provided with some form of direct instruction. If the first phase is conducted outside of class as suggested, instruction can be provided by having students watch a video that introduces operons (e.g., Andersen, n.d.) and by reading the relevant pages in their textbook. In addition, they can perform an introductory activity with an operon simulation (e.g., PhET Team, 2013) to further equip them with a basic understanding of operon structure and function.

The second phase of the activity begins the next time the students meet for class. The structures and functions of the trp and lac operons can be briefly reviewed through discussion and probing questions. I typically use a computer simulation projected on a screen to focus the discussion. There are a number of simulations available online, including the PhET simulation mentioned earlier (PhET Team, 2013). Alternatively, students may work in small groups to construct physical models of the operons with paper cutouts, Playdoh, or other suitable material while the instructor provides guidance and probes for understanding. Instructional modeling kits are also available from suppliers (e.g., Bullock, n.d.). The focused discussion takes ~20 minutes, and the modeling activity takes ~40 minutes. The instructor should be sure that students have a basic understanding of operons and of the differences between repressible and inducible operons before proceeding further with the second phase.

Following the review, students work in small groups to complete the problem-solving exercises in Tables 1 and 2, which challenge them to predict the behavior of operons in mutant strains under varying conditions. These exercises require students to reason in a manner similar to the way Jacob and Monod did as they developed their model of the lac operon. Depending on the ability level of the students, this may take 40–50 minutes. I use the following questions to focus students' attention during this phase of the activty: "How does E. coli 'know' how to behave in such a seemingly puposeful manner? How does it 'know' whether tryptophan and lactose are there or not? How does it 'know' that the glucose is gone? How does it respond to changes in its environment in order to acquire the necessary energy and raw materials to maintain homeostasis?" To answer these questions, students must understand how a network of molecules linked by negative feedback loops can respond "intelligently" to fluctuating environmental conditions.

Once students have completed both the trp and lac tables, they may formatively assess some of their predictions on the lac operon table (Table 2) by experimenting again with the PhET simulation. For example, if a problem calls for a mutation in the repressor protein, students would simply leave the gene for the repressor out of the simulation and observe the effect. Answers to the problems in Tables 1 and 2 are provided for the instructor.

In the third and final phase of the activity, students reflect on the Big Ideas. They work in their groups to answer questions that require them to think about how the functioning, or malfunctioning, of operons is related to broad themes in biology (see Appendix). Alternatively, these questions can be assigned for homework; however, the ideal is to have students work in groups in class, where the instructor can foster an environment that promotes student discussion and collaboration. In addition, the instructor can circulate during discussions and formatively assess the students' responses.

Upon completion of phase 3, the instructor should lead a class discussion to wrap up the activity, reviewing parts 2 and 3 and revisiting the focus questions. As a culminating assessment, students can be given one or two new scenarios to evaluate for each operon. By manipulating the variables in the operons as was mentioned above, it is possible to construct a large number of additional scenarios, not found in Tables 1 and 2, that can be used for assessment.

Relating Operons to Themes of Biology

In addition to challenging students to reason with models and promoting understanding of the function of the trp and lac operons, this activity promotes reflection on the Big Ideas in the AP Biology Framework (College Board, 2012). Specifically, the activity can be used to teach students about the importance of exchanging matter and energy with the environment and the role of feedback in maintaining homeostasis; the evolutionary consequences of regulating or not regulating gene expression; that genes store information about the successes of an organism's ancestors; and that organisms have complex emergent properties due to interactions between their constituent parts.

Maintenance of Homeostasis

An organism is best understood as a dynamic process, like a flame, rather than as an object. A constant flow of matter and energy is required to maintain order in the face of the disordering tendencies of the second law of thermodynamics. The ability to maintain a dynamic balance in the face of these disordering tendencies is called "homeostasis." To help maintain homeostasis, bacteria like E. coli organize their genes in operons that enable rapid responses to challenges posed by disordering fluctuations in the environment. The ability of a collection of essentially "dumb" elements like genes and proteins to act apparently intelligently and maintain homeostasis results from the process called "negative feedback." Negative feedback determines whether the operon is on or off, on the basis of the output of the process itself, and is responsible for the consistent patterns of behavior generated by organisms whereby they maintain various aspects of their metabolism within specific ranges. In negative feedback, a small change causes a response from the system that tends to counteract the change, thereby contributing to the stability of the system. For example, if your body temperature rises too high, a behavior like sweating is initiated to cool the body and bring the temperature back down toward the normal level.

Negative feedback requires a sensor, a control mechanism, and an effector. The sensor monitors the current level of some variable and reports to the control mechanism. The control mechanism "evaluates" the current level of the variable and directs the activity of the effector. In a negative feedback loop, the effector takes an action that tends to counteract changes, maintaining stability. An easily understood example of a homeostatic mechanism is a home heating system. The temperature of a home is maintained during cold weather by a heater that is controlled by negative feedback. The thermostat contains a sensor that monitors the temperature and a control mechanism that compares the temperature to a set point, then activates or deactivates an effector (the heater) to cause changes in the system. The result is a stable temperature in the home.

The action of an operon is under the control of the repressor, which serves a role similar to that of the control mechanism in a thermostat. The sensor is the allosteric site on the repressor where the corepressor or inducer binds, changing the conformation of the repressor. The effector is the DNA binding site of the repressor protein. Figure 3 illustrates a feedback loop for the trp operon.

Figure 3.

Feedback in the trp operon. The diamond-shaped box represents a point in the feedback cycle where a decision is made. If tryptophan levels are low (A), the trp operon is on synthesizing the enzymes necessary to make tryptophan, and the tryptophan level in the cell rises. However, if tryptophan is already present at high levels in the cell (B), a tryptophan molecule will bind to the allosteric site of the trp repressor protein, altering its shape such that the repressor then binds to the operator blocking transcription of the genes for making more tryptophan. The level of tryptophan in the cell will gradually fall as the cell uses it to manufacture proteins.

Figure 3.

By default, the trp repressor is inactive and does not bind to DNA. If tryptophan is not readily available in the environment, there will be none to bind to the inactive repressor molecule. The operon will be on, causing tryptophan to be synthesized and the level to rise. By contrast, if tryptophan is readily available, the sensor (repressor's allosteric site) will detect its presence and the control mechanism (repressor protein) will activate the effector (alter the conformation of the repressor's DNA binding site), causing the repressor to bind to the operator and shut down production until the level of tryptophan in the cell falls. A similar diagram can be drawn for the lac operon if the CAP volume control is omitted. Including CAP would make the diagram a little more complicated, but it can be done. Drawing a diagram based on Figure 4 that also includes CAP is suggested as an extension activity for more advanced students (see Figure 5).

Figure 4.

Feedback in the lac operon. The diamond-shaped box represents a point in the feedback cycle where a decision is made. If lactose levels are high (A), an allolactose molecule will bind to the allosteric site of the lac repressor protein, altering its shape such that the repressor no longer binds to the operator, freeing the promoter so that RNA polymerase can bind. In this case, the lac operon is on, synthesizing the enzymes necessary to hydrolyze lactose, and the lactose level in the cell falls. However, if there is no lactose in the cell (B), the repressor protein, free of allolactose, will bind to the operator and block transcription. The operon is off. The lactose level will rise again only if the host consumes lactose.

Figure 4.

Figure 5.

The lac operon with CAP. In addition to the operator–repressor interaction, which serves as an on–off switch for the lac operon, a second controlling element, the catabolite activator binding site, interacts with the catabolite activator protein (CAP) to serve as a volume control, dialing the transcription rate up or down, depending on the glucose level. When both glucose and lactose are available, E. coli exhibits a preference for glucose. Glucose is used as the primary energy source, and the lac operon genes are transcribed only at a low level. Once the glucose is all used up, the rate of transcription of lac enzymes increases and the cell begins to use the lactose at a higher rate. This increase in the rate of transcription is a result of CAP binding to the CAP binding site just upstream from the promoter and bending the DNA so that RNA polymerase binds more tightly to the promoter.

Figure 5.

It is essential for students to understand how organisms employ negative feedback in maintaining homeostasis and regulating gene expression. However, the terminology used in this activity (sensor, effector, and control mechanism) may add a cumbersome level of detail for some students. At the discretion of instructors, these terms and Figure 3, Figure 4, and Figure 5 can be omitted from the discussion and students can still be taught the basics of negative feedback.

Evolution & Information

Proper maintenance of homeostasis has short-term implications for the individual organism because the failure to maintain homeostasis will likely result in the death of the organism. But there are also long-term implications for the species. Organisms that perish because they cannot maintain homeostasis will not reproduce, and their genes will be lost from the gene pool, changing the genetic make-up of the population.

The evolutionary consequences of the proper functioning of the trp and lac operons become apparent when one considers the costs and benefits of transcribing the genes in the operon under varying conditions. For example, if there is no tryptophan present in the environment, it makes good sense for the operon to be on and making tryptophan, because tryptophan is a basic building block for many of the proteins the cell needs to function. However, if there is already an abundance of tryptophan present in the environment, it then makes good sense for the bacterial cell to take advantage of the windfall, rather than waste energy and resources producing a molecule that is readily available at little or no cost to the cell. The manufacture of five proteins necessary for tryptophan synthesis, followed by the synthesis of the tryptophan itself, would consume a considerable amount of energy and raw materials that could be used more productively for another purpose. The failure to use resources wisely in an environment where they are limited will diminish reproductive success, so organisms with mutations that prevent them from regulating gene expression will likely be eliminated by natural selection.

A similar cost–benefit analysis can be done for the lac operon. The default state of the lac operon is off, so none of the enzymes for processing lactose are synthesized unless the potential energy source, lactose, is actually present. In terms of the organism's survival in the face of competition for limited resources, this makes perfect sense because it prevents the cell from wasting energy and resources producing an abundance of enzymes that would not contribute to the survival and reproductive success of the organism.

From the perspective of the evolutionary history of E. coli and its hosts, the fact that the genetic elements described in these operons even exist is a consequence of the reproductive successes of their ancestors' struggle to survive in their environments. The genes that selectively allow E. coli to make tryptophan for biosynthesis and use lactose as an energy source are products of natural selection acting on the ancestors of E. coli. In addition, the bacteria reside in the digestive tracts of animals, so the dietary choices of their hosts also played a role in the evolution of the operons, given that tryptophan and lactose must have been periodically present in the hosts' diets before there could have been selection for the operons. The operons represent stored information about the history of successes as previous generations struggled to survive under varying conditions in their hosts' guts.

Interactions & Emergent Properties

Living systems are organized hierarchically, and novel properties emerge at each higher level as a result of interactions between parts at the lower level. The emergence of seemingly purposeful, higher-level properties from "dumb" elements interacting at a lower level is a difficult concept for students to grasp (Resnick, 1996; Chi, 2005). In particular, organized behavior emerging from a collection of randomly moving molecules may seem very unlikely to students whose mental models of molecular behavior go no further than the billiard-ball model of kinetic theory. Unlike the molecules of a gas, which move randomly, bouncing off one another in a chaotic swirl of activity, the molecules of cells are constrained, to a degree, in their interactions by weak, transient, intermolecular attractions between their surfaces. Molecular surfaces may have polar, electrically charged, or hydrophobic regions. Rather than randomly colliding and bouncing off, interactions between surfaces of proteins and other molecules provide a mechanism that causes molecules to aggregate in specific ways and result in emergent behaviors. As Jacob (1976, p. 304) observed, "A whole series of biological structures – polymers, membranes and intracellular organelles – thus have their own internal logic, a logic which is not exactly that of the three-dimensional crystals, but very little different. All these structures can exercise a chemical function only through their surface."

Prior to the 20th-century revolution that led to molecular biology, the apparently purposeful behavior of living systems gave rise to various forms of vitalism – the belief that explanations of living things required some principle above and beyond the laws of physics and chemistry. Such notions have been abandoned by biologists who understand that systems of interacting elements can produce apparently purposeful behavior as a result of the constraints placed on their interactions by specific rules – in this case, the rules of chemistry, negative feedback, and natural selection. Most biological functions arise from interactions between many protein components (Alberts, 1998; Hartwell et al., 1999). The focus of cell biology has shifted away from a preoccupation with single protein molecules toward a focus on molecular complexes that perform the functions of the cell (Hartwell et al., 1999). The molecular complexes spontaneously self-assemble from their individual protein components as a result of their surface interactions.

This trend toward thinking of systems of molecules interacting to generate complex behaviors is simply a continuation and extension of ideas Jacob and Monod developed to explain the lac operon. Contemplating a diagram of cellular metabolism, Monod remarked, "Even if at each step each enzyme carried out its job perfectly, the sum of their activities could only be chaos were they not somehow interlocked so as to form a coherent system" (1971, p. 62). The interlocked network of molecules in the trp and lac operons form coherent systems that result in responsive and seemingly intelligent behavior on the part of E. coli.

Conclusion

By using operons to teach students to reason with models of complex systems and understand broad themes, we equip them with powerful skills and ideas that form a solid foundation for their future learning in biology. These skills and ideas have broad application in biology, but they also potentially have application in other areas. Constructs, such as negative feedback and natural selection, that are used to explain changing and self-organizing systems constitute what Ohlsson (1993) refers to as "abstract schemas," which encode the structure of discourse rather than its content. Their abstract nature allows for the possibility of cross-domain transfer. For example, the schema for negative feedback was developed originally in the context of specific engineering problems and was later found to have application in biology. Similarly, natural selection was developed to explain the origin of adaptations in organisms but has subsequently been applied to the development of the immune and nervous systems, computer programming, and artificial intelligence. Mastering abstract schemas enables students to develop into mature thinkers with powerful minds who can imagine solutions to the world's future problems.

References

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Alberts, B. (1998). The cell as a collection of protein machines: preparing the next generation of molecular biologists. Cell, 92, 291–294.

Bullock, C. (n.d.). Lac operon: Turning on your genes activity kit. Ward's Science catalog no. 4738400. Available at http://www.wardsci.com.

Chi, M.T.H. (2005). Commonsense conceptions of emergent processes: why some misconceptions are robust. Journal of the Learning Sciences, 14, 161–199.

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Jacob, F. (1976). The Logic of Life: A History of Heredity. New York, NY: Vintage.

Jacob, F. (2011). The birth of the operon. Science, 332, 767.

Monod, J. (1971). Chance and Necessity. New York, NY: Knopf.

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Appendix: Questions Related to the Big Ideas

What is homeostasis? Why is it necessary for organisms to maintain homeostasis?

The steady-state physiological condition of the body. A failure to maintain homeostasis can be caused by disease or extreme conditions and can result in the death of the organism.
What is negative feedback, and why is it important to the maintenance of homeostasis?

A form of regulation in which accumulation of an end product of a process slows the process. Negative feedback enables organisms to maintain physiological variables within normal ranges.

A description of the function of the trp operon is provided below, along with a flow diagram that illustrates the regulation of the operon through negative feedback control (Figure 3). As you read the explanation, identify the parts of the negative feedback control mechanism and answer questions 3 through 7. Refer to the diagram as needed.

The trp Operon

The action of the trp operon is under the control of a repressor protein, which regulates the activity of the operon. By default, the repressor protein has a conformation that prevents it from binding to the operator. However, when the corepressor (tryptophan) is present and binds to an allosteric site on the repressor, the result is a change in the conformation of the repressor. The conformation of the repressor protein determines whether the repressor binds to the DNA at the operator site or not. Thus, the presence or absence of tryptophan, and the resulting conformation of the repressor, determines whether the operon is on (transcribing genes) or off (not transcribing genes).

3. What is the name of the environmental variable whose level must be regulated in order to maintain homeostasis?

Tryptophan level
4. Identify the part of the trp operon system that acts as the control mechanism in the feedback loop.

trp repressor protein
5. What part of the trp operon system is the sensor that detects the level of the environmental variable and communicates that information to the control mechanism?

Allosteric site on the repressor where tryptophan binds as a corepressor
6. What part of the trp operon system is the effector that enables the control mechanism to influence the level of the environmental variable?

Site where the repressor protein binds to (or does not bind to) the DNA
7. Write an If… then… else statement that accurately reflects the decision process determining whether the operon is on or off.

If the tryptophan level is low, then the operon is on and tryptophan is synthesized, else the operon is off and tryptophan is not synthesized.

A description of the function of the lac operon is provided below, along with a flow diagram that illustrates the regulation of the operon through negative feedback control (Figure 4). As you read the explanation, identify the parts of the negative feedback control mechanism and answer questions 8 through 12. Refer to the diagram as needed.

The lac Operon

The action of the lac operon is under the control of a repressor protein, which regulates the activity of the operon. By default, the repressor protein binds to the operator site of the lac operon and prevents transcription of the structural genes. When the inducer (allolactose) binds to the allosteric site on the repressor, the result is a change in the conformation of the repressor that releases it from the operator site and exposes the promoter to RNA polymerase. The conformation of the repressor protein determines whether the repressor binds to the DNA at the operator site or not. Thus, the presence or absence of lactose, and the resulting conformation of the repressor, determines whether the operon is on (transcribing genes) or off (not transcribing genes).

8. What is the name of the environmental variable whose level is regulated by the lac operon?

Lactose level
9. Identify the part of the lac operon system that acts as the control mechanism in the feedback loop.

lac repressor protein
10. What part of the lac operon system is the sensor that detects the level of the environmental variable and communicates that information to the control mechanism?

Allosteric site where allolactose binds as an inducer
11. What part of the lac operon system is the effector that enables the control mechanism to influence the level of the environmental variable?

Site where the repressor protein binds to (or does not bind to) the DNA
12. Write an If… then… else statement that accurately reflects the decision process determining whether the operon is on or off.

If the lactose level is high, then the operon is on and enzymes for processing lactose are synthesized, else the operon is off and the enzymes are not synthesized.
13. What would be the long-term consequence for the organism of a mutation in the operator or the repressor that resulted in constitutive (continuous) production of the lac operon enzymes?

Cost–benefit analysis: Production of lac operon enzymes at times when no lactose is present would waste energy and resources. Faced with competition for resources, the organism would likely perish without leaving offspring.
14. Suppose a bacterial cell had a mutation in the trp R gene that resulted in a repressor that was unable to bind the corepressor tryptophan. What would be the effect on the functioning of the trp operon? How would such a bacterium, with this mutation, living in an environment with limited resources, be able to compete with other bacteria lacking the mutation in the trp R gene? Explain.

Cost–benefit analysis: The operon would remain on (i.e., the enzymes would be produced constitutively) even when tryptophan is readily available in the environment. Faced with competition for resources, the organism would likely perish without leaving offspring.

Read the passage below and answer question 15.

Molecules of a gas move randomly bouncing off one another in a chaotic swirl of activity. The molecules of cells also move randomly about, but when they bump into one another they are constrained to a degree in their interactions by weak, transient, intermolecular attractions between their surfaces. These interactions result from partial or full charges on the molecules, or from hydrophobic interactions. These interactions provide a mechanism that causes molecules to aggregate in specific ways and results in emergent structures and behaviors. Consider the way that random interactions between water molecules and amphipathic phospholipids result in the formation of a lipid bilayer. François Jacob (1976, p. 304) observed, "A whole series of biological structures – polymers, membranes and intracellular organelles – thus have their own internal logic, a logic which is not exactly that of the three-dimensional crystals, but very little different. All these structures can exercise a chemical function only through their surface."

15. Consider either the interactions between the repressor, corepressor, and the DNA at the operator site in the trp operon, or the repressor, inducer, and the DNA at the operator site in the lac operon, and explain how random motions of, and dynamic interactions between, these molecules contribute to the coordinated behavior of E. coli as it maintains homeostasis. Explain how these interactions illustrate what François Jacob meant by the quote in the passage above.

Jacob is making an analogy between the interaction of ions or molecules forming the ordered structure of a crystal and the ordered behavior of the molecules within cells. The molecules of cells are constrained, to a degree, in their interactions by weak, transient, intermolecular attractions between their surfaces. The overall result when small molecules, proteins, and DNA interact through these surfaces is the ordered behavior of the system of molecules. This is somewhat analogous to ions or molecules interacting through their charges to form the ordered structure of a crystal.
16. Textbooks often characterize DNA as a molecule that controls cellular activities. Based on your understanding of the functioning of the trp and lac operons, is it accurate to characterize DNA as the controller or conductor of the bacterial cell's responses to the presence of tryptophan and lactose in its environment? Explain.

Rather than being a conductor of the cell's activities, DNA is a repository of hereditary information which is essentially inert without its interactions with proteins and RNA molecules that enable the hereditary information to be expressed. Ordered expression of genes arises from random movements and interactions between DNA, proteins, and small molecules.
17. Modify the feedback flow diagram for the lac operon (Figure 4) to include CAP.

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2015