Studying Gene Expression: Database Searches and Promoter Fusions to Investigate Transcriptional Regulation in Bacteria

Abstract

A laboratory project was designed to illustrate how to search biological databases and utilize the information provided by these resources to investigate transcriptional regulation in Escherichia coli. The students searched several databases (NCBI Genomes, RegulonDB and EcoCyc) to learn about gene function, regulation, and the organization of transcriptional units. A fluorometer and GFP promoter fusions were used to obtain fluorescence data and measure changes in transcriptional activity. The class designed and performed experiments to investigate the regulation of genes necessary for biosynthesis of amino acids and how expression is affected by environmental signals and transcriptional regulators. Assessment data showed that this activity enhanced students’ knowledge of databases, reporter genes and transcriptional regulation.

INTRODUCTION

Genomics and bioinformatics have been described as useful topics for project-based learning because students carry out database searches and enhance their computer skills while investigating genome structure and gene function (8). Advances in the field of microbial genomics have generated bioinformatics resources that are freely accessible to undergraduate educators. Databases such as EcoCyc (5) and RegulonDB (11) provide valuable data on gene structure, function and regulation of model organisms, specifically Escherichia coli (E. coli). These resources present an ideal opportunity to introduce students to biological databases and to incorporate bioinformatics into the biology curriculum.

The aim of the laboratory project presented in this report was to use publicly available information on gene structure and regulation, together with green fluorescent protein (GFP) promoter fusions, to illustrate changes in gene expression in response to different cellular and environmental conditions. GFP is a powerful tool to monitor gene expression and GFP reporter libraries have been employed to follow the transcriptional activities of hundreds of genes in the E. coli genome (16). Therefore, GFP reporter constructs present a valuable tool to teach undergraduate students about transcriptional regulation. Several reports have described laboratory exercises and projects using GFP as a tool to teach about the location of molecules in cells, cloning and protein purification (13, 15); however, only a few laboratory activities have used GFP to study transcriptional regulation (6).

Using the green fluorescence protein as a reporter to monitor gene expression presents several advantages when studying the principles of gene regulation in an undergraduate laboratory setting. First, transcriptional activity can be quantified in one step, by measuring fluorescence. This procedure avoids the cell lysis and addition of substrates that are necessary when performing beta galactosidase assays. Secondly, GFP reporter constructs are available for most genes in the E. coli genome, facilitating the development of laboratory exercises that can investigate the transcriptional regulation of several genes at the time. Finally, the analysis of fluorescence data presents an ideal opportunity for students to strengthen their quantitative and data interpretation skills.

Two genes involved in amino acid metabolism, gltB and serA, were selected to develop this activity. These genes were chosen due to their relevance in amino acid biosynthesis and their transcriptional regulation in response to several cellular and environmental cues. The gltB gene encodes the large subunit of glutamate synthase, an enzyme that catalyzes the following reactions:

$$\text{L-glutamate}+\text{H}2\text{O}+{\text{NADP}}^{+}\leftrightarrows 2-\text{ketoglutarate}+\text{ammonia}+\text{NADPH}$$

$$2\hspace{0.17em}\text{L-glutamate}+{\text{NADP}}^{+}\leftrightarrows \text{L-glutamine}+2-\text{ketoglutarate}+\text{NADPH}$$

Glutamate synthase uses ammonia as a nitrogen source for the generation of L-glutamate from alpha-ketoglutarate. In addition, glutamate synthase catalyzes the single-step conversion of L-glutamine and alpha-ketoglutarate into two molecules of L-glutamate. In doing so, it simultaneously operates as the major source of L-glutamate for the cell and as a key step in ammonia assimilation during nitrogen-limited growth (9). D-3-phosphoglycerate dehydrogenase, the enzyme encoded by serA, catalyzes the following reactions:

$$2-\text{hydroxyglutarate}+{\text{NAD}}^{+}\rightleftarrows 2-\text{oxoglutarate}+\text{NADH}+{\text{H}}^{+}$$

$$3-\text{phosphoglycerate}+{\text{NAD}}^{+}\leftrightarrows 3-\text{phospho-hydroxypyruvate}+\text{NADH}+{\text{H}}^{+}$$

These reactions constitute the first committed step in the biosynthesis of L-serine from glucose in bacterial cells (14).

This activity was successfully implemented and field tested as a three-week laboratory project on gene expression and transcriptional regulation. The students that participated in the project were juniors and seniors taking upper level biology courses, specifically, microbiology and biochemistry. The laboratory module emphasized database searching skills, exploration of gene structure and quantitative analysis of gene expression. In the first week, the students searched public databases and learned about the structure and regulation of gltB and serA. Simultaneously, they were trained on how to operate a fluorometer and utilize fluorescence measurements to quantify changes in gene expression. During the second week, the students proposed research questions on transcriptional regulation, formulated hypotheses, and designed experiments involving GFP reporter constructs and fluorescence measurements to test their assumptions. In the final week of the project, the designed experiments were implemented. Each laboratory team wrote a final report presenting the experimental outcomes, showing the analysis of fluorescence data and discussing how the results supported or refuted the proposed hypotheses.

Intended audience

For effective participation in this project, students should have completed a semester of genetics and a semester of cellular biology. The laboratory exercise described in this report was designed for upper level microbiology and biochemistry courses. The students should have knowledge of the scientific method, the principles of gene structure, amino acid function and metabolism.

Knowledge of the role of transcription factors in gene regulation and how these proteins mediate changes in gene expression in response to external stimuli is essential for the successful implementation of this laboratory exercise. Instructors should start this activity by reviewing the mechanisms that cells use to regulate gene expression. Undergraduates often have misconceptions about transcriptional regulators and the way in which small molecules affect gene expression. For example, some students think that transcription factors only bind to one site in the genome and regulate one gene at the time. Other common mistakes include: 1) thinking that metabolites directly bind DNA and that such binding can control the activity of a gene, and 2) assuming that binding of a small molecule to a transcription factor will always result in either activation or repression. When reviewing transcriptional regulation, it is important to emphasize that the control of gene expression in response to environmental stimuli is mediated by transcription factors and that small metabolites bind to these proteins to stimulate or repress their activity. The instructor should explain that transcription factors can bind to one or more specific DNA sequences in a genome and that these proteins can regulate the activity of one or many genes. Furthermore, it is useful to review examples of transcriptional regulation before starting the laboratory activity. The class should discuss the concept of allosteric regulation and classic examples of positive and negative control of gene expression such as the arabinose and lactose operons in E. coli.

After the discussion on transcriptional regulation, the class should transition into the bioinformatics part of the laboratory activity. A good way to switch to this topic is by asking the students how the availability of genome sequences can be used to learn about gene regulation. This question often prompts the class to talk about computational analyses for the identification promoter sequences and DNA binding sites for transcription factors. The instructor can take advantage of this topic to introduce biological databases and the type of information stored in these resources. The discussion can be finalized by briefly describing the databases that will be explored in this exercise, and the type of information that can be obtained by searching these resources.

Learning objectives

Upon completion of this project, students will be able to:

1. Explain the principles linking the activity of transcription factors to regulation of gene expression in response to different stimuli.

2. Search public databases to obtain information about transcription factors, regulatory sequences and the regulation of gene expression.

3. Define the term “reporter gene” and list several examples of genes used in reporter assays.

4. Describe the organization of a “reporter gene” construct and discuss its application to the study of transcriptional regulation.

5. Design and perform experiments involving gene reporters and fluorescence to measure gene expression.

6. Analyze fluorescence data and discuss whether it supports or refutes a given hypothesis.

PROCEDURE

Materials and methods

E. coli strains

The strains needed for this project are described in Table 1 (strains constructed for this activity are available from the author upon request). Alternatively, strains such as MG1655 containing plasmid pUA66 (a map of pUA66 is provided in Appendix XII) and its derivatives containing the gltB and serA promoter GFP fusions are commercially available. The wild type E. coli K12 strain MG1655 and its isogenic lrp, nac and argR mutants can be obtained through the Coli Genetic Stock Center (CGSC) at Yale University; argR mutants can also be obtained from their original source (10). Mutants obtained from the CGSC have a kanamycin resistance cassette that must be removed before transforming the cells with pUA66 and its derivates; these vectors also contain a Kan^R gene as a selective marker. The removal of the antibiotic resistance cassettes had been described by Dastenko and Wanner (3). The authors are in process of providing the Kanamycin sensitive strains described in this article to the CGSC.

TABLE 1.

E. coli strains used in this work; all strains are isogenic with E. coli K-12. The “P” next to gene names on the first column stands for promoter. Strain LP1000 is E. coli W3110 with a Δlac-169 mutation; W3110 is a common laboratory strain of E. coli with a genetic background that is very similar to MG1655 (10).

Strain	Description	Reference or Source
E. coli MG1655	Wild type E. coli K-12	CGSC at Yale University
E. coli MG1655 (pUA66)	Wild type E. coli K-12 containing pUA66 with promoterless GFP	Zaslaver et.al.(16)
E. coli LP1050 (pUA66)	LP1000 ΔargR containing pUA66	This work
E. coli MG1655 [pUA66 (PgltB-GFP)]	Wild type E. coli K-12 containing pUA66 with a gltB-GFP promoter fusion	Zaslaver et.al.(16)
E. coli MG1655 [pUA66 (PserA-GFP)]	Wild type E. coli K-12 containing pUA66 with a serA-GFP promoter fusion	Zaslaver et.al.(16)
E. coli MG1655 (Δlrp) [pUA66 (PgltB-GFP)]	lrp deletion mutant containing pUA66 with a gltB-GFP promoter fusion	This work
E. coli MG1655 (Δlrp) [pUA66 (PserA-GFP)]	lrp deletion mutant containing pUA66 with a serA-GFP promoter fusion	This work
E. coli MG1655 (Δnac) [pUA66 (PgltB-GFP)]	nac deletion mutant containing pUA66 with a gltB-GFP promoter fusion	This work
E. coli MG1655 (Δnac) [pUA66 (PserA-GFP)]	nac deletion mutant containing pUA66 with a serA-GFP promoter fusion	This work
E. coli LP1050 (ΔargR) [pUA66 (PgltB-GFP)]	argR deletion mutant containing pUA66 with a gltB-GFP promoter fusion	This work
E. coli LP1050 (ΔargR) [pUA66 (PserA-GFP)]	argR deletion mutant containing pUA66 with a serA-GFP promoter fusion	This work

Materials and equipment

The main instrumentation necessary for this project are a fluorometer and a spectrophotometer to measure absorbance and monitor microbial growth. Many fluorometers have absorbance modules that easily allow simultaneous measurements of absorbance and fluorescence. The data presented in this report was obtained using a Turner Biosystems Modulus Fluorometer Microtiter Plate Reader with fluoresecence and absorbance capabilities. The green fluorescence protein present in pUA66 has an excitation of wavelength of 481 nm and emission maxima of 507 nm (2). Fluorescence measurements were taken using the fluoromoter’s Blue Kit (Ex 490 nm, Em 510 – 570 nm); absorbance at 600 nm was used to determine the growth of the cultures. Other equipment needed for this project includes a shaker incubator, pipettors and sterile culture tubes (glass tubes or plastic sterile falcon tubes).

Reagents and microbial media

Amino acids (hydrochlorides or disodium salt forms) were purchased from Sigma Chemicals. M9, yeast extract and Luria Bertani medium were prepared as described by Sambrook (12). The M9 medium used for most of the exercises described in this paper contained 0.4% glucose as a carbon source and was supplemented with 0.025% yeast extract. Other versions of this medium used for student’s projects were M9 + 0.4% lactose + 0.025% yeast extract and M9 + 0.4% glycerol + 0.025% yeast extract.

Experimental set up for promoter activity assays

The activity of the gltB and serA promoters was monitored using GFP promoter fusions and taking fluorescence and absorbance measurements of E. coli cultures in mid-exponential phase. Promoter activity assays were set up as follows: 0.5 ml of a fresh overnight culture of the E. coli [ pUA66 (gltBp-GFP)] was added to 2.5 ml of LB broth and 2.5 ml of M9 + 0.4% glucose medium supplemented with 0.025% yeast extract. The absorbance of the cultures after this dilution was 0.120. The cells were transferred to an incubator shaker and grown by shaking at 250 rpm and 37°C until they reached O.D. 600 equal to 0.4 (approximately two hours). Fluorescence and absorbance measurements were taken to verify microbial growth and assess promoter activity.

When studying the effect of different substances on promoter activity, the assay conditions were modified to accommodate the volume of the added reagents. For example, when investigating the effect of leucine on promoter activity, 0.1 ml of 100 mM leucine were added to a tube containing 0.5 ml of overnight cell culture and 2.4 ml of M9 (0.4% glucose + 0.025% yeast extract) medium.

Quantification of promoter activity by normalized fluorescence

Fluorescence measurements tend to be noisy because growth media and microbial cells have background readings when analyzed in a fluorometer. In addition, differences in growth rate and number of cells influence fluorescence measurements. To correct for these artifacts, a control strain containing the pUA66 vector with a promoter-less GFP was used as reference in every assay (16). Furthermore, raw fluorescence measurements were normalized to account for differences in microbial growth and cell numbers (16). Calculations were performed as follows:

$$\begin{array}{l}\mathit{Normalized}\hspace{0.17em}\mathit{Fluorescense}\hspace{0.17em}(\mathit{NF})=\\ \hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\mathit{Raw}\hspace{0.17em}\mathit{Fluorescence}\hspace{0.17em}/\mathit{Absorbance}\end{array}$$

$$\begin{array}{l}\mathit{Corrected}\hspace{0.17em}\mathit{Fluorescence}=\\ \hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}(\mathit{NF}\hspace{0.17em}\mathit{fluorescence}\hspace{0.17em}\mathit{in}\hspace{0.17em}\mathit{experimental}\hspace{0.17em}\mathit{sample}-\\ \hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}\mathit{NF}\hspace{0.17em}\mathit{fluorescence}\hspace{0.17em}\mathit{in}\hspace{0.17em}\mathit{pUA66}\hspace{0.17em}\mathit{control}\hspace{0.17em}\mathit{strain})\end{array}$$

Corrected fluorescence was used as the measurement for comparing promoter activities.

Week one: bioinformatics tools to understand transcriptional regulation

Laboratory periods were three hours long and students always worked in groups of three. During the first week of the project, the students spent two hours doing bioinformatics and one hour learning how to operate the fluorometer and analyze fluorescence data. At the beginning of the first lab period, the class was asked to dilute overnight cultures of E. coli MG1655 [pUA66(gltBp-GFP)] into fresh M9 (minimal medium) and LB (rich medium) broth; each culture was diluted in the medium used for overnight growth. The cells were placed in the incubator for two hours before taking fluorescence and absorbance measurements. A sample of each culture (0.150 ml) was taken and saved to have a record of the initial absorbance and fluorescence of the cultures.

While waiting for the cells to grow, the class was presented with a set of questions to explore their knowledge of gene expression and regulation (Appendix I). The class was given 10 minutes to answer the questions. A discussion on gene reporters, promoter fusions and their usefulness in monitoring gene expression followed. This dialogue allowed the students to understand the relevance of the strains that they were growing, the choice of growth media and the overall goal of the three-week project that they had begun.

Subsequently, the class started to work on the bioinformatics part of the project. This part consisted of searching publicly available databases to gain knowledge about the function of gltB and serA, their transcriptional units and regulation. The bioinformatics module included searches using the following resources:

• EcoCyc: http://www.ecocyc.org/

• Regulon DB: http://regulondb.ccg.unam.mx/

• NCBI Genome: http://www.ncbi.nlm.nih.gov/sites/entrez?db=Genome&itool=toolbar

Since gltB and serA are genes present in the E. coli genome, the Encyclopedia of Escherichia coli K-12 Genes and Metabolism (EcoCyc) was used as the primary resource to learn about the function of these genes, their structure, role in metabolism and transcriptional regulation. EcoCyc is a bioinformatics database that describes the genome and the biochemical machinery of E. coli K-12 MG1655. The goal of this database is to describe the molecular catalog of the E. coli cell, as well as the functions of each of its molecular parts, to facilitate a system-level understanding of E. coli. EcoCyc is an electronic reference source for E. coli biologists, and for biologists who work with related microorganisms (5). The students continued their search by exploring RegulonDB, a database that models the mechanisms of transcriptional regulation in E. coli. RegulonDB provides information regarding genes in transcription units, operons and regulatory networks. RegulonDB staff curates the transcriptional information presented in EcoCyc; both databases contain the same information regarding gene regulation in E. coli. RegulonDB provides alternative formats to view information on transcriptional regulation. In addition, this database contains regulatory network tools that are not available through EcoCyc. Both resources provide video tutorials that can be accessed through the following URLs: http://regulondb.ccg.unam.mx/html/Demos.jsp (RegulonDB), http://ecocyc.org/webinar.shtml and http://ecocyc.org/samples.shtml for EcoCyc. Detailed instructions on how to use these databases are provided in Appendix XI.

A worksheet was provided for the students to complete as they worked through the bioinformatics exercise (Appendix II). Some of the guide questions in the worksheet can be answered using either EcoCyc or RegulonDB – it is up to instructor to specify which database should be used to answer a particular question.

Week one: using fluorescence to monitor changes in gene expression

In the second part of the laboratory period, the students were trained on using a fluorometer to obtain absorbance and fluorescence measurements. Overnight E. coli cultures were retrieved from the incubator, diluted (0.5 ml in 2.5 ml of growth medium) and placed in microtiter plates (0.150 ml) to obtain initial absorbance and fluorescence measurements. The diluted cell cultures were placed in the incubator; fluorescence and absorbance measurements were taken again after two hours of incubation. The students filled out a worksheet reporting the absorbance and fluorescence of their cultures (Appendix III). The data collected during this exercise was reviewed and discussed during the third week of the laboratory project.

Week two: designing an experiment

After completing the bioinformatics part of the project, the students use their knowledge of the transcriptional control of gltB and serA to design an experiment and address a question about the regulation of these genes. Each laboratory group was asked to choose one of the transcription factors that control gltB or serA and search NCBI PubMed for papers describing its function and relevance to cellular metabolism. Although the regulation databases (EcoCyc and RegulonDB) provide literature references to support their data, a supplementary PubMed search was added to this exercise as a way to enhance the student’s ability to examine the primary scientific literature. The purpose of this search was to get the class thinking about potential research questions concerning transcription factors and how they mediate gene expression in response to environmental cues. The students were told that besides the reagents available during the first week, they could have access to the following strains and materials:

• ♦ 100 mM solutions of 20 common amino acids

• ♦ Incubators at 30°C, 37°C and 42°C

• ♦ M9 Growth media at pH 5, 6, 7, 8 and 9

• ♦ M9 Growth medium with lactose as a carbon source

• ♦ M9 Growth medium with glycerol as a carbon source

• ♦ M9 Growth medium with low ammonium concentration (50% less than the original medium)

• ♦ M9 growth medium high ammonium concentration (100% more than the original medium)

• ♦ 100 mM ammonium chloride

• ♦ Bacterial strains: E. coli LP1050 ΔargR (gltBp-GFP); E. coli LP1050 ΔargR (serAp-GFP); E. coli BW25113 Δnac (serAp-GFP); E. coli BW25113 Δnac (gltBp-GFP); E. coli MG1655 Δlrp (gltBp-GFP); E. coli MG1655 Δlrp (serAp-GFP).

At the end of this laboratory period, each group turned in a worksheet describing their literature search, hypothesis to be tested, experimental design and a list of strains and reagents needed to carry out the proposed experiment the following week (Appendix IV).

Week three: setting up the experiment and collecting data

In the first part of this laboratory period, the students set up their experiments and placed their cultures in the incubator for two hours. The remaining class time was used to review the analysis of the fluorescence data collected during the first week. The instructor reviewed the purpose of taking absorbance and fluorescence measurements, highlighted the concept of normalized fluorescence and its significance. In addition, initial and final normalized fluorescence calculations were compared; the advantages and potential limitations of this experimental technique were discussed. Afterwards, the class was prompted to discuss the following question: Does the growth medium have an effect in the activity of the gltB promoter? Support your answer using fluorescence data obtained by your lab group.

Example of instructor’s data

In order to illustrate how to operate the fluorometer and use fluorescence measurements to study transcriptional regulation, the activity of the gltB promoter was evaluated in cells grown in rich (LB) and minimal (M9 + 0.4% glucose + 0.025% yeast extract) media. Since gltB encodes a protein involved in amino acid biosynthesis, promoter activity is expected to be higher in minimal medium when compared to rich medium. Rich medium contains amino acids and abundant carbon and nitrogen sources that allow bacteria to grow without having to activate the biosynthetic pathways involving gltB. As shown in Fig. 1, cells grown on M9 minimal medium have three times more promoter activity than the cells grown on LB. These data illustrates the usefulness of the promoter GFP reporter system to distinguish differences in promoter activity and transcriptional regulation.

FIGURE 1.

Experimental data showing the activity of the gltB promoter in E.coli cells grown on rich and minimal media. The data shown is an average of six replicas. A paired t-test was performed to evaluate the significance of the results; the data were significant and had a p value of less than 0.000002. Fluorescence measurements were obtained after cultures were grown for two hours and had reached an OD600 of 0.4.

Example of student’s data

Laboratory groups worked on studying a variety factors that could influence the transcriptional regulation of gltB and serA. Several projects investigated the effects of transcription factors on promoter regulation, while others looked at the effects of environmental conditions on transcriptional activity. Figure 2 illustrates data collected by students who were studying the activity of the gltB and serA promoters in wild type cells and mutants lacking Lrp, a transcription factor that regulates both genes. The experimental data supported the function of Lrp as a transcriptional activator of both promoters.

FIGURE 2.

An Example of experimental data obtained by the students. The activity of serA and gltB promoters was studied in wild type cells and cells lacking Lrp, a transcriptional regulator of both genes. Cells were grown on minimal medium to observe optimal promoter activity for gltB and serA. The student’s data confirmed that Lrp is a transcriptional activator of gltB and serA. The data shown is the average of three replicas; error bars indicate the standard error of the mean values.

DISCUSSION

Field testing

This laboratory module was field tested with biology majors taking upper division microbiology and biochemistry courses at Hamline University during the Spring and Fall of 2009. A total of 41 students participated in the laboratory project and the assessment of this activity.

Evidence of student learning

Several methods were used to evaluate student learning during the course of the laboratory activity. The scores of pre- and post-tests were compared to assess objectives 1, 3, 4 and 6. Seven data interpretation questions and an exercise on database searching were included in the course’s final exam. These scores were used to evaluate objectives 3 and 6 (see Appendix V and VI). The student’s ability to design experiments involving gene reporters and to analyze fluorescence data (objectives 5 and 6) was assessed by grading weekly worksheets and group laboratory reports at the end of the three-week exercise. An example of student’s laboratory report is presented in Appendix VII.

During the Spring of 2009, results of the pre- and post-tests showed that students’ scores increased from 62% to 83% after the completion of the three-week lab activity (Table 2). Similarly, students’ scores increased from 53% to 72% after completion of this activity in the Fall of 2009 (Table 2). Analysis of students’ responses to individual questions in the pre- and post-tests revealed a significant increase in the percentage of students who correctly answered specific questions regarding gene reporters and biological databases. In contrast, the scores for most of the review questions on concepts related to transcriptional regulation were comparable for the majority of the students (Table 3). The microbiology students fared better than the biochemistry class in the pre- and post-tests. This might be due to fact that most students taking microbiology were seniors, while the biochemistry class mostly had juniors. Senior students have had more coursework and exposure to the concepts and techniques necessary to understand the regulation of gene expression. Therefore, the background knowledge of the senior students could have allowed them to do better in the pre- and post-tests.

TABLE 2.

Results of pre- and post-tests used to evaluate student learning after the completion of the laboratory project. The test’s results were analyzed by using a paired-two tail t-test. The statistics showed that the data were significant with a p value of less than 0.0001 (p < 0.0001) at 95% confidence interval.

Course	Average Score
	Pre-Test	Post-Test	p-value
Microbiology (Spring 2009) (n=16)	62%	83%	< 0.0001
Biochemistry (Fall 2009) (n=25)	53%	72%	< 0.0001

TABLE 3.

Analysis of student’s responses to individual questions in pre- and post-tests; the percentage of correct answers shows the number of students who provided the right answer to a given question. The tests had a multiple choice format; some questions were slightly modified (re-phrased) to fit into Table 3. A detailed copy of the exam (questions and alternatives to choose from) is shown in Appendix V. Review questions are denoted by an R.

Question	Percent of Correct Answers
	Pre-Test Spring 09 / Fall 09	Post-Test Spring 09 / Fall 09
What does a reporter construct should have to be effective as a tool for monitoring transcriptional regulation?	30% / 24%	68% / 70%
Why are reporter genes useful to monitor gene expression?	60% / 56%	83% / 80%
List three examples of reporter genes	5% / 8%	62 % / 55%
What type of information is available in the Ecocyc database?	3%/ 4%	75% / 28%
Which databases provide detailed information about transcription factors and the genes that they regulate?	75% / 60%	83% / 68%
Your Biology professor created a library of reporter constructs in which different pieces of DNA were cloned upstream of a promoter-less beta galactosidase gene. What are these “reporter” constructs meant to report?	40% / 56%	82% / 88%
R How does a repressor inhibit the synthesis of mRNA?	100% / 100%	100% / 96%
R What should happen to promoter activity In the presence of a transcriptional activator?	83% / 92%	85% / 100%
R Why do cellular metabolites influence the activity of transcription factors?	83% / 76%	95% / 96%
R Select all the statements that are true about transcription factors.	70% / 64%	80% / 72%
R What should happen to promoter activity in the presence of a transcriptional repressor?	85% / 100%	85% / 100%

The average score in the database search exercise and the fluorescence data analysis section of the final exam was 87% (Spring 2009) and 85% (Fall 2009). These results illustrate that the students learned how to effectively search databases containing information about transcription factors, interpret fluorescence data and relate it to a given experimental scenario.

In summary, the assessment results suggest that this laboratory activity allowed the students to review the concepts governing gene expression while learning about the usefulness of reporter genes and the information available in biological databases.

Potential modifications

Variations of this activity include the incorporation of parts of the project as stand-alone laboratory exercises in other courses. For example, RegulonDB and EcoCyc contain information on the regulation of every gene present in the E. coli genome; therefore, the bioinformatics exercises can be used as an independent laboratory assignment in genetics and molecular biology courses. Plasmids containing GFP fused to most E. coli promoters are commercially available; thus, the system described in this article can be employed to introduce undergraduates to the use of fluorescence as a tool to monitor gene expression in bacteria. The fluorescence measurements generated during this laboratory exercise provide an excellent dataset for statistical analyses. The students should be encouraged to do a t-test or other statistics to evaluate the accuracy of their data. Additional projects might include studying the regulation of genes involved in nitrogen metabolism, utilization of alternativecarbon sources and stress responses.

Alternatively, if your institution does not have a fluorometer, the activities described in this report can be carried out using lacZ as a reporter gene and quantifying beta galactosidase activity with a spectrophotometer. Strains containing lacZ promoter fusions might be available from several research groups that have used this gene as a reporter to study transcriptional regulation (1, 4, 7). Finally, since this laboratory activity requires that students design their own experiments, instruction on the scientific method might be necessary if the participants have not designed experiments in previous courses. Adding a section on the scientific method and its application to the study of gene regulation will make this laboratory exercise more appropriate for students who are working on investigative projects for the first time. Several curriculum resources for teaching the scientific method are available through the MicrobeLibrary (http://www.microbelibrary.org).

SUPPLEMENTAL MATERIALS

Appendix I: Questions used to explore student’s knowledge of gene expression and regulation

Appendix II: Bioinformatics Worksheet

Appendix IIa: Bioinformatics Worksheet- Answer Key

Appendix III: Absorbance and Fluorescence Worksheet

Appendix IV: Literature Search and Experimental Design

Appendix V: Pre- Test and Post-Test

Appendix Va: Answer Key for Pre- and Post-Test

Appendix VI: Bioinformatics Search Exercise and Exam Questions

AppendixVII: Example of Student’s Laboratory Report

AppendixVIII: Rubric borrowed from LabWrite and modified to grade laboratory

Appendix IX: Laboratory Prep

Appendix X: Student’s Handout

Appendix XI: Instructions for Database Searching

Appendix XII: Map for Plasmid pUA66