Article Isolation and Genetic Analysis of an Environmental Bacteriophage: A 10-Session Laboratory Series in Molecular Virology
Ryan P. Williamson Brent T. Barker Hamidou Drammeh Jefferson Scott Joseph Lin*
From the Department of Biology at Sonoma State University, Rohnert Park, California 94928
Abstract Bacterial viruses, otherwise known as bacteriophage (or phage), are some of the most abundant viruses found in the environment. They can be easily isolated from water or soil and are ideal for use in laboratory classrooms due to their ease of culture and inherent safety. Here, we describe a series of 10 laboratory exercises where students collect, isolate, and purify the genome of an environmental phage. Once the genome has been extracted,
students then clone a fragment of their isolated phage genome into a plasmid and analyze its sequence to identify the phage in their original isolate. These exercises have been carefully designed to apply foundational concepts that will expose students to basic skills in microbiolC 2014 by The ogy, molecular biology, and bioinformatics. V International Union of Biochemistry and Molecular Biology, 42(6):480–485, 2014.
Keywords: molecular virology; bacteriophage; laboratory exercises
Introduction Foundational laboratory skills in molecular biology are an important component to any biology curriculum; however, applying these techniques to problem-based learning poses several challenges due to the technical skill required [1]. Most modern molecular biology laboratory courses are built around disconnected, short, “cookbook”-style exercises designed to fit within a given timeframe. More recently, there has been a trend toward more inquiry-based learning [2, 3]. Labs designed with an undetermined result focus on guided investigation by students, rather than following explicitly described protocols [4]. These strategies encourage students to develop and use critical thinking skills by allowing them to encounter and overcome problems as they arise. Here, we describe a 10-session laboratory series in a molecular virology course where each session builds upon the previous, culminating in the identification of an unknown bacteriophage from an environmental sample. The course is designed for upper-division undergraduate biology majors, using a broad array of microbiological and molecu-
*Address for correspondence to: Department of Biology at Sonoma State University, Rohnert Park, California 94928, USA. E-mail: [email protected] Received 28 July 2014; Revised 25 August 2014; Accepted 7 September 2014 DOI 10.1002/bmb.20829 Published online 21 October 2014 in Wiley Online Library (wileyonlinelibrary.com)
480
lar techniques. By allowing students to follow the thread of inquiry from start to finish over an extended period of time, they become more invested in each session’s results, as they will have an impact on their future experiments in the course [2, 3]. The genuinely unknown aspect of the bacteriophage sample the students collect also fosters greater curiosity and improved engagement in laboratory exercises. Laboratory courses in a specialized field such as virology are uncommon at most undergraduate institutions; however, viruses are an ideal system to apply techniques central to molecular biology. For practical reasons, such as ease of culturing and student safety, environmental bacteriophage are good model viruses for use in a laboratory classroom [5, 6]. They can easily be found in ponds, streams, and even simply in the soil [7]. Bacteriophage not only have a wide range of habitats, they are also biochemically and molecularly diverse, consisting of a wide range of protein capsids, lipid envelopes (or the absence thereof), and genomic composition. For this series of exercises, an environmental bacteriophage sample will be collected and propagated using Escherichia coli in the laboratory. Its genome will then be purified and characterized using various nucleases. Finally a genomic fragment will be ligated into a plasmid and the sequence analyzed to identify the phage isolated from the original sample (summarized in Fig. 1).
Course Development This series of exercises was first incorporated into the existing Virology course (Biol 383) at Sonoma State
Biochemistry and Molecular Biology Education
University in the spring semester of 2012. Previously, Virology was taught as a lecture (3 hr/wk) and discussion (1 hr/ wk) course, however to provide a more “hands on” approach to the study of viruses, this lab series was developed in place of the discussion. The course is intended for upper division Biology or Biochemistry majors, therefore, lower division Molecular and Cell Biology (w/lab), a year of General Chemistry (w/lab), and one semester of Organic Chemistry serve as prerequisites for the course. Each lab section contains 16 students creating 8 lab groups of two students each. One of the major objectives of this course is to have students apply concepts from prerequisite courses to a specific discipline within biology, such as virology in this case. During the typical prerequisite courses listed above, students learned fundamental lab skills such as the use of micropipettes, aseptic microbiological techniques, DNA restriction enzyme analysis, and agarose gel electrophoresis [8]. Students also have a basic understanding of laboratory calculations such as determining molar concentrations and serial dilutions. Another attribute of this lab series is that the cost is minimal and it does not require specialized instrumentation beyond typical microbiological reagents and supplies and introductory molecular biology and biochemistry equipment.
Student Learning Objectives Expectations for participation are described in the syllabus and reiterated during the laboratory orientation on the first day. In brief, student attendance and participation are required and essential for success in the course. One of the most important factors for success is having a solid understanding of basic molecular and cellular processes that are covered in the prerequisite “Molecular and Cell Biology” course. Viruses have evolved to take advantage of cellular machinery to propagate, so to understand viruses, students must understand normal cellular processes. The students’ initial introduction to the molecular and cell biology techniques from the prerequisite course was of the typical “cookbook” variety. The goal of this series of exercises is to continue the development of these foundational techniques common to modern molecular biology labs and demonstrate how they are applied to address a specific question. The end result will hopefully prepare students for multiple future scientific pursuits as well as satisfy the biotech industry’s expectations for entry-level technicians [9]. Learning objectives for the lab portion of the course are as follows:
Quantitative Skills Perform calculations to make laboratory solutions. Perform calculations to determine viral titers. Calculate unknown DNA fragment sizes using a M.W. standard.
Technical Laboratory Skills Make common laboratory solutions. Pour bacterial agar plates.
Williamson et al.
FIG 1
Project overview. Flow chart describing the major steps of this series of exercises starting from bacteriophage sample collection to bacteriophage genome analysis.
Perform serial dilutions and plaque assays. Isolate and amplify phage from a single plaque. Biochemically purify nucleic acids. Perform restriction digests and agarose gel electrophoresis.
Bioinformatics Skills Understand the basics of DNA sequence assembly and analysis. Perform BLAST searches. Navigate some of the sequence databases at the NCBI web site. The goals for each lab session and an outline of the procedures were presented at the beginning of each session. Written lab reports were then due the following session. Writing a report after lab required students to not only know the procedure, but also be able to understand and justify each step.
Laboratory Exercises The following exercises were designed for 10 laboratory sessions of 3 hr each. At Sonoma State University, the exercises are performed each week over the course of a semester. Each lab session includes a short introduction by the instructor and a brief discussion of the goals and procedures for the lab period. Complete lab handouts containing background, reagents, and protocols for each session can be found in the supplement. Handouts were kept brief to
481
Biochemistry and Molecular Biology Education of their plates, and assess whether their serial dilution was performed successfully. To ensure that only one phage strain is carried forward, a single plaque is selected, extracted, and another serial dilution is performed. If there are multiple plaque morphologies, students are encouraged to re-plate at least two, to observe the difference after replating. Because some student samples may not have any plaques, students can use plaques from other groups, especially from those groups that contain plates with multiple plaque morphologies. Students again serially dilute the reisolated phage and plate them with top agar and fresh E. coli on TSA plates.
Session 4: Recalculate Titer and Precipitate Phage
FIG 2
Plaque assay. Initial phage-containing samples were amplified in liquid culture with host E. coli for 24 hr. Samples were then centrifuged, filtered, given fresh host bacteria, and plated in agar. Plates were then incubated overnight. Note the various plaque morphologies.
encourage students to research and justify in their lab report why specific reagents were used.
Session 1: Preparing Laboratory Reagents and Collecting Phage Samples The first session has two goals: preparing reagents needed throughout the semester and acquiring an environmental bacteriophage sample. For the former, students are given desired volumes and concentrations; they are instructed to calculate the amount of reagent needed and then prepare them. Although the reagents and media are being autoclaved, students go off to collect an environmental bacteriophage sample from somewhere on campus. Any nonchlorinated water source or moist area can be a promising source of bacteriophage, such as a pond or creek. After students have obtained their sample they attempt to amplify the bacteriophage by adding Tryptic Soy Broth (TSB) and E. coli. The samples are then incubated overnight at 37 C and refrigerated until the next lab period.
Session 2: Serial Dilutions and Plating After a brief introduction to the concepts of viral plaques and plaque-forming units, debris and bacteria are filtered out of the amplified phage cultures using a 0.2 mm filter. Students then serially dilute this original stock and plate them with top agar and fresh E. coli on TSA plates. After incubating the plates overnight, the plates are refrigerated until the next session (Typical results shown in Fig. 2).
Session 3: Calculate Phage Titers and Re-isolate a Single Plaque Students are taught to calculate a titer (plaque-formingunit (PFU)/mL) to estimate the viral titer based on several
482
A titer (PFU/mL) is again determined this time on a single phage strain. The viral particles are then extracted from the agar from the plate with the highest concentration of phage by soaking in SM buffer. The phage is then precipitated from the SM buffer with PEG and high-speed centrifugation. The phage particle pellets are then stored overnight at 4 C.
Session 5: Extract Phage Genome With the viral particles isolated, the genome is then extracted from the bacteriophage using SDS and NaOH followed by a phenol/chloroform/isoamyl alcohol (25:24:1) extraction. The nucleic acid is then precipitated with EtOH and allowed to air dry. The purified genome is then stored until the next week.
Session 6: Identify the Nature of the Genome and Ligate It into a Plasmid Phage can have a genome of DNA or RNA, either double or single stranded, and carrying each one forward in this experiment requires different techniques. Because it requires no additional work, only samples that have dsDNA genomes are carried forward. If time allows, an additional lab period could be devoted to include ssDNA or RNA viruses using reverse transcriptase and/or random primer PCR. To determine the nature of the genome, four reactions are prepared. The genome is cut with RNAse, DNAse, the restriction endonuclease HaeIII (GG/CC), or left uncut as a control. These reactions are then resolved using agarose gel electrophoresis (sample results shown in Fig. 3). The remaining sample of the HaeIII digests is then heat denatured and ligated overnight into pBluescript II vector that has been cut with EcoRV. Importantly, HaeIII and EcoRV both leave “blunt” ends after digestion. Again, any groups whose samples did not contain dsDNA can obtain a sample from another group.
Session 7: Bacterial Transformation and Screen The ligated genomic fragments are now transformed into competent E. coli. The transformed E. coli are plated onto ampicillin plates containing IPTG and X-Gal to allow screening for inserts. Plates are then incubated overnight. The next day, students are asked to come in for a few
Laboratory Series in Molecular Virology Using Bacteriophage
FIG 4 FIG 3
Characterization of phage genome. Purified phage genome was incubated with RNAse, DNAse, the restriction enzyme HaeIII (GG/CC), or left untreated. (a) Shows a typical dsDNA genome whereas (b) shows an RNA genome. Note that the RNA genome in (b) is significantly degraded as precautions to avoid RNAse contamination were not in place.
minutes to examine their plates containing blue and white colonies and to start six small liquid cultures from only colonies with plasmids carrying an insert (white colonies). These cultures are then incubated overnight and stored until the next session.
Session 8: Plasmid Isolation and Restriction Digest The plasmids from the six E. coli cultures are purified using a standard alkaline lysis protocol. The plasmids are then cut with BamHI and HindIII to remove the plasmid insert (a fragment of the phage genome). The restriction digests are then resolved using agarose gel electrophoresis (sample results are shown in Fig. 4). Clones with an insert >500 bp but
Ryan P. Williamson Brent T. Barker Hamidou Drammeh Jefferson Scott Joseph Lin*
From the Department of Biology at Sonoma State University, Rohnert Park, California 94928
Abstract Bacterial viruses, otherwise known as bacteriophage (or phage), are some of the most abundant viruses found in the environment. They can be easily isolated from water or soil and are ideal for use in laboratory classrooms due to their ease of culture and inherent safety. Here, we describe a series of 10 laboratory exercises where students collect, isolate, and purify the genome of an environmental phage. Once the genome has been extracted,
students then clone a fragment of their isolated phage genome into a plasmid and analyze its sequence to identify the phage in their original isolate. These exercises have been carefully designed to apply foundational concepts that will expose students to basic skills in microbiolC 2014 by The ogy, molecular biology, and bioinformatics. V International Union of Biochemistry and Molecular Biology, 42(6):480–485, 2014.
Keywords: molecular virology; bacteriophage; laboratory exercises
Introduction Foundational laboratory skills in molecular biology are an important component to any biology curriculum; however, applying these techniques to problem-based learning poses several challenges due to the technical skill required [1]. Most modern molecular biology laboratory courses are built around disconnected, short, “cookbook”-style exercises designed to fit within a given timeframe. More recently, there has been a trend toward more inquiry-based learning [2, 3]. Labs designed with an undetermined result focus on guided investigation by students, rather than following explicitly described protocols [4]. These strategies encourage students to develop and use critical thinking skills by allowing them to encounter and overcome problems as they arise. Here, we describe a 10-session laboratory series in a molecular virology course where each session builds upon the previous, culminating in the identification of an unknown bacteriophage from an environmental sample. The course is designed for upper-division undergraduate biology majors, using a broad array of microbiological and molecu-
*Address for correspondence to: Department of Biology at Sonoma State University, Rohnert Park, California 94928, USA. E-mail: [email protected] Received 28 July 2014; Revised 25 August 2014; Accepted 7 September 2014 DOI 10.1002/bmb.20829 Published online 21 October 2014 in Wiley Online Library (wileyonlinelibrary.com)
480
lar techniques. By allowing students to follow the thread of inquiry from start to finish over an extended period of time, they become more invested in each session’s results, as they will have an impact on their future experiments in the course [2, 3]. The genuinely unknown aspect of the bacteriophage sample the students collect also fosters greater curiosity and improved engagement in laboratory exercises. Laboratory courses in a specialized field such as virology are uncommon at most undergraduate institutions; however, viruses are an ideal system to apply techniques central to molecular biology. For practical reasons, such as ease of culturing and student safety, environmental bacteriophage are good model viruses for use in a laboratory classroom [5, 6]. They can easily be found in ponds, streams, and even simply in the soil [7]. Bacteriophage not only have a wide range of habitats, they are also biochemically and molecularly diverse, consisting of a wide range of protein capsids, lipid envelopes (or the absence thereof), and genomic composition. For this series of exercises, an environmental bacteriophage sample will be collected and propagated using Escherichia coli in the laboratory. Its genome will then be purified and characterized using various nucleases. Finally a genomic fragment will be ligated into a plasmid and the sequence analyzed to identify the phage isolated from the original sample (summarized in Fig. 1).
Course Development This series of exercises was first incorporated into the existing Virology course (Biol 383) at Sonoma State
Biochemistry and Molecular Biology Education
University in the spring semester of 2012. Previously, Virology was taught as a lecture (3 hr/wk) and discussion (1 hr/ wk) course, however to provide a more “hands on” approach to the study of viruses, this lab series was developed in place of the discussion. The course is intended for upper division Biology or Biochemistry majors, therefore, lower division Molecular and Cell Biology (w/lab), a year of General Chemistry (w/lab), and one semester of Organic Chemistry serve as prerequisites for the course. Each lab section contains 16 students creating 8 lab groups of two students each. One of the major objectives of this course is to have students apply concepts from prerequisite courses to a specific discipline within biology, such as virology in this case. During the typical prerequisite courses listed above, students learned fundamental lab skills such as the use of micropipettes, aseptic microbiological techniques, DNA restriction enzyme analysis, and agarose gel electrophoresis [8]. Students also have a basic understanding of laboratory calculations such as determining molar concentrations and serial dilutions. Another attribute of this lab series is that the cost is minimal and it does not require specialized instrumentation beyond typical microbiological reagents and supplies and introductory molecular biology and biochemistry equipment.
Student Learning Objectives Expectations for participation are described in the syllabus and reiterated during the laboratory orientation on the first day. In brief, student attendance and participation are required and essential for success in the course. One of the most important factors for success is having a solid understanding of basic molecular and cellular processes that are covered in the prerequisite “Molecular and Cell Biology” course. Viruses have evolved to take advantage of cellular machinery to propagate, so to understand viruses, students must understand normal cellular processes. The students’ initial introduction to the molecular and cell biology techniques from the prerequisite course was of the typical “cookbook” variety. The goal of this series of exercises is to continue the development of these foundational techniques common to modern molecular biology labs and demonstrate how they are applied to address a specific question. The end result will hopefully prepare students for multiple future scientific pursuits as well as satisfy the biotech industry’s expectations for entry-level technicians [9]. Learning objectives for the lab portion of the course are as follows:
Quantitative Skills Perform calculations to make laboratory solutions. Perform calculations to determine viral titers. Calculate unknown DNA fragment sizes using a M.W. standard.
Technical Laboratory Skills Make common laboratory solutions. Pour bacterial agar plates.
Williamson et al.
FIG 1
Project overview. Flow chart describing the major steps of this series of exercises starting from bacteriophage sample collection to bacteriophage genome analysis.
Perform serial dilutions and plaque assays. Isolate and amplify phage from a single plaque. Biochemically purify nucleic acids. Perform restriction digests and agarose gel electrophoresis.
Bioinformatics Skills Understand the basics of DNA sequence assembly and analysis. Perform BLAST searches. Navigate some of the sequence databases at the NCBI web site. The goals for each lab session and an outline of the procedures were presented at the beginning of each session. Written lab reports were then due the following session. Writing a report after lab required students to not only know the procedure, but also be able to understand and justify each step.
Laboratory Exercises The following exercises were designed for 10 laboratory sessions of 3 hr each. At Sonoma State University, the exercises are performed each week over the course of a semester. Each lab session includes a short introduction by the instructor and a brief discussion of the goals and procedures for the lab period. Complete lab handouts containing background, reagents, and protocols for each session can be found in the supplement. Handouts were kept brief to
481
Biochemistry and Molecular Biology Education of their plates, and assess whether their serial dilution was performed successfully. To ensure that only one phage strain is carried forward, a single plaque is selected, extracted, and another serial dilution is performed. If there are multiple plaque morphologies, students are encouraged to re-plate at least two, to observe the difference after replating. Because some student samples may not have any plaques, students can use plaques from other groups, especially from those groups that contain plates with multiple plaque morphologies. Students again serially dilute the reisolated phage and plate them with top agar and fresh E. coli on TSA plates.
Session 4: Recalculate Titer and Precipitate Phage
FIG 2
Plaque assay. Initial phage-containing samples were amplified in liquid culture with host E. coli for 24 hr. Samples were then centrifuged, filtered, given fresh host bacteria, and plated in agar. Plates were then incubated overnight. Note the various plaque morphologies.
encourage students to research and justify in their lab report why specific reagents were used.
Session 1: Preparing Laboratory Reagents and Collecting Phage Samples The first session has two goals: preparing reagents needed throughout the semester and acquiring an environmental bacteriophage sample. For the former, students are given desired volumes and concentrations; they are instructed to calculate the amount of reagent needed and then prepare them. Although the reagents and media are being autoclaved, students go off to collect an environmental bacteriophage sample from somewhere on campus. Any nonchlorinated water source or moist area can be a promising source of bacteriophage, such as a pond or creek. After students have obtained their sample they attempt to amplify the bacteriophage by adding Tryptic Soy Broth (TSB) and E. coli. The samples are then incubated overnight at 37 C and refrigerated until the next lab period.
Session 2: Serial Dilutions and Plating After a brief introduction to the concepts of viral plaques and plaque-forming units, debris and bacteria are filtered out of the amplified phage cultures using a 0.2 mm filter. Students then serially dilute this original stock and plate them with top agar and fresh E. coli on TSA plates. After incubating the plates overnight, the plates are refrigerated until the next session (Typical results shown in Fig. 2).
Session 3: Calculate Phage Titers and Re-isolate a Single Plaque Students are taught to calculate a titer (plaque-formingunit (PFU)/mL) to estimate the viral titer based on several
482
A titer (PFU/mL) is again determined this time on a single phage strain. The viral particles are then extracted from the agar from the plate with the highest concentration of phage by soaking in SM buffer. The phage is then precipitated from the SM buffer with PEG and high-speed centrifugation. The phage particle pellets are then stored overnight at 4 C.
Session 5: Extract Phage Genome With the viral particles isolated, the genome is then extracted from the bacteriophage using SDS and NaOH followed by a phenol/chloroform/isoamyl alcohol (25:24:1) extraction. The nucleic acid is then precipitated with EtOH and allowed to air dry. The purified genome is then stored until the next week.
Session 6: Identify the Nature of the Genome and Ligate It into a Plasmid Phage can have a genome of DNA or RNA, either double or single stranded, and carrying each one forward in this experiment requires different techniques. Because it requires no additional work, only samples that have dsDNA genomes are carried forward. If time allows, an additional lab period could be devoted to include ssDNA or RNA viruses using reverse transcriptase and/or random primer PCR. To determine the nature of the genome, four reactions are prepared. The genome is cut with RNAse, DNAse, the restriction endonuclease HaeIII (GG/CC), or left uncut as a control. These reactions are then resolved using agarose gel electrophoresis (sample results shown in Fig. 3). The remaining sample of the HaeIII digests is then heat denatured and ligated overnight into pBluescript II vector that has been cut with EcoRV. Importantly, HaeIII and EcoRV both leave “blunt” ends after digestion. Again, any groups whose samples did not contain dsDNA can obtain a sample from another group.
Session 7: Bacterial Transformation and Screen The ligated genomic fragments are now transformed into competent E. coli. The transformed E. coli are plated onto ampicillin plates containing IPTG and X-Gal to allow screening for inserts. Plates are then incubated overnight. The next day, students are asked to come in for a few
Laboratory Series in Molecular Virology Using Bacteriophage
FIG 4 FIG 3
Characterization of phage genome. Purified phage genome was incubated with RNAse, DNAse, the restriction enzyme HaeIII (GG/CC), or left untreated. (a) Shows a typical dsDNA genome whereas (b) shows an RNA genome. Note that the RNA genome in (b) is significantly degraded as precautions to avoid RNAse contamination were not in place.
minutes to examine their plates containing blue and white colonies and to start six small liquid cultures from only colonies with plasmids carrying an insert (white colonies). These cultures are then incubated overnight and stored until the next session.
Session 8: Plasmid Isolation and Restriction Digest The plasmids from the six E. coli cultures are purified using a standard alkaline lysis protocol. The plasmids are then cut with BamHI and HindIII to remove the plasmid insert (a fragment of the phage genome). The restriction digests are then resolved using agarose gel electrophoresis (sample results are shown in Fig. 4). Clones with an insert >500 bp but
