Fire at Serum Institute’s Manjri plant in Pune; 10 fire tenders at the spot While the plant produces multiple vaccines, the facility affected by fire did not have any production work on at the time of the incident, said the police By Shalaka Shinde UPDATED ON JAN 21, 2021 05:00 PM IST A fire broke out at the Manjri plant Serum Institute of India (SII), Pune, on Thursday afternoon. While the plant produces multiple vaccines, the facility affected by fire did not have any production work on at the time of the incident, said the police. The cause of fire is being investigated, said fire brigade officials. The call was received at 2.50pm after which 10 fire tenders and at least two tankers were rushed to the spot, said Pune mayor Murlidhar Mohol. Later, chief minister Uddhav Thackeray’s office tweeted, “CM Uddhav Balasaheb Thackeray is in touch with the Pune Municipal Commissioner and is taking complete on-ground updates. He has directed the state machinery to coordinate and ensure that the situation is under control.” Deputy CM Ajit Pawar said in a statement, “Efforts to douse fire at Serum Institute of India are on at a war footing. The city and district administration have been activated in helping the operation. Directives have been issued to the Pune Police commissioner for detailed investigation into the incident. Following the incident, concerns have been expressed in the country and abroad. I have been told that the plant where Covid-19 vaccine is being manufactured is safe. At present, our focus is to douse the fire and minimize the damage.” Pune Police commissioner Amitabh Gupta confirmed that the facility had nothing to do with Covid vaccine. “The primary information was that there’s a fire on the fourth and fifth floor of the building in the Manjri plant of the institute. No production was going on at the moment. Fortunately, there is no casualty. The first count of people has been done and a second count is going on. We are hoping that the fire will be brought under control in the next hour.” SII official Vivek Pradhan also confirmed that the plant wasn’t involved in Covishied production.“This plant has nothing to do with Covishield. The Covishield plant is at least a km away from this building. Only raw material was inside this building. Nobody is trapped there. There were not more than 5-6 people in that space,” he said. SII CEO Adar Poonawalla tweeted, “Thank you everyone for your concern and prayers. So far the most important thing is that there have been no lives lost or major injuries due to the fire, despite a few floors being destroyed.” Local MLA Chetan Tupe said after reaching the spot, “I checked with fire brigade officials and they told me no casualty has been reported so far. The fire broke out at M SEZ-3, a newly constructed building. It is a part of Special Economic Zone developed by the Serum Institute. No vaccine production was going on there.”

Singapore-based startup Breathonix announced that it has developed a breath test that can detect Covid-19 within a minute. Breathonix CEO Zhunan Jia (left) and chief operating officer Fang Du / Photo credit: Breathonix Breathonix CEO Zhunan Jia (left) and chief operating officer Fang Du / Photo credit: Breathonix The company says that the test has achieved more than 90% accuracy in a clinical trial involving 180 patients. It also detected with a 95% specificity, which means it could identify those without the disease in 95 out of 100 cases. Breathonix, which was spun off from the National University of Singapore, has a track record in detecting lung cancer and tuberculosis by analyzing volatile molecules in exhaled breath. The company is backed by early-stage venture capital firm Antler, which in April invested a total of up to US$500,000 in startups that are tackling the Covid-19 outbreak. How Does It Work? In this, a machine learning software is used which subsequently analyses the VOC profile and generates the result in less than a minute. Further as explained by Breathonix CEO Dr Jia Zhunan, VOCs are consistently produced by various biochemical reactions in human cells. Dr Jia said, “Different diseases cause specific changes to the compounds, resulting in detectable changes in a person’s breath profile. As such, VOCs can be measured as markers for diseases like COVID-19,”. While talking about its working, the firm’s chief operating officer, Du Fang said, “the system’s disposable mouthpiece has a one-way valve and a saliva trap which prevents inhalation and any saliva from entering the machine”. “This makes cross-contamination unlikely,” he added. So far, this NUS start-up Breathonix technology has achieved more than 90 percent accuracy in a clinical trial which involved 180 patients. This technology, “offers a fast and convenient solution to identify COVID-19 infection”, according to the university. Roadmap For Phase 3 Currently, Singapore is working on the roadmap for the phase three of easing of safety measures under the circuit breaker, or semi-lockdown, to control the spread of COVID-19. Further, the city-state will continue to remain in Disease Outbreak Response System Condition (DORSCON) Orange “for the time being”, the highest alert, according to the COVID-19 multi-ministry task force. From November, a group of up to 20 people would be allowed to take part in activities like walking, cycling and kayaking tours, which is better from the current maximum size of 10, said by the Singapore Tourism Board on Monday. Under safety measures, travel agencies and tour operators are allowed to resume on-site operations from October 19. So far, six new COVID-19 cases were reported on Tuesday, which included four imported ones, taking the total infections to 57,921.

Modern techniques in Biotech The discovery that genes are made up of DNA and can be isolated, copied and manipulated has led to a new era of modern biotechnology. New Zealand has many applications for modern biotechnologies. DNA extraction A forensic scientist extracting DNA. Humans have been manipulating living things for thousands of years. Examples of early biotechnologies include domesticating plants and animals and then selectively breeding them for specific characteristics. Find out more about ancient biotechnology. Modern biotechnologies involve making useful products from whole organisms or parts of organisms, such as molecules, cells, tissues and organs. Recent developments in biotechnology include genetically modified plants and animals, cell therapies and nanotechnology. These products are not in everyday use but may be of benefit to us in the future. Applications in biotechnology Key applications of biotechnology include: DNA profiling – for further information see the article DNA profiling DNA cloning – for further information see the article DNA cloning transgenesis genome analysis stem cells and tissue engineering – for further information see the article Stem cells xenotransplantation – for further information see the article Xenotransplantation. Meeting human needs and demands Dog breeds The selection of particular traits - or selective breeding - is how people have bred dogs for specific purposes. For example the 'sausage dog' - or dachshund were said to have been bred specifically for hunting badgers - their short, long body suited to entering badger holes. Working dog breeds from The New Student’s Reference Work published in 1914. Biotechnologies have an important role in meeting human needs and demands in medicine, agriculture, forensics, bioremediation, biocontrol and biosecurity. Medicine Gene modification or transgenesis are used to produce therapeutic human proteins in cells or whole organisms. The cell or organism used depends upon how large and complex the protein is. For example, human insulin, a small protein used to treat diabetes, is made in genetically engineered bacteria, whereas large, more complex proteins like hormones or antibodies are made in mammalian cells or transgenic animals. Antibiotics and vaccines are products of microorganisms that are used to treat disease. Modern biotechnologies involve manipulating vaccines so they are more effective or can be delivered by different routes. Gene therapy technologies are being developed to treat diseases like cancer, Parkinson’s disease and cystic fibrosis. In New Zealand, gene therapy is being used as a way to target and kill cancer cells with fewer side effects. Xenotransplantation is the transplanting of cells, tissue or organs from one species into another. In New Zealand, cells from a unique, virus-free population of pigs are being used to treat people with type 1 diabetes. Find out more about this in the article Pig cell transplants. For further information see the article, Xenotransplantation. Agriculture Plants and animals can be improved by selectively breeding for particular traits or by genetic modification. Beneficial traits can be identified visually or by DNA profiling. For example, farmers may want plants with herbicide or insect resistance, tolerance to different growing environments or improved storage, or they may want livestock with better meat and wool or resistance to disease. Forensics DNA profiling is used in forensic analysis to identify DNA samples at a crime scene or to determine parentage. For further information see the article, Forensics and DNA. Bioremediation Organisms or parts of organisms can be used to clean up pollution in soil, water or air. In New Zealand, bioremediation has been suggested as an effective way of removing the toxin DDT from the soil. Lace bug In 2009 the lace bug, Gargaphia decoris, was approved as a biocontrol agent for woolly nightshade in New Zealand. Woolly nightshade is an invasive weed that is well established in the North Island. For further information see article, Public acceptance of bioremediation to address New Zealand’s DDT problem. Biocontrol and biosecurity Biocontrol is when one organism is used to control the levels of another. Biocontrol methods are being used in New Zealand to control invasive plants and insects. For further information, see the article, Biocontrol. Biocontrol is also being explored as an option to control numbers of possums in New Zealand. For further information, see the article, Biological control of possums. Impacts of biotechnology on society Biotechnologies use organisms or part of organisms to make a product to meet a specific human need. This raises social and ethical issues that are important to discuss. For further information, see the article, Impacts of biotechnology on society.

Mammalian cell tissue culture techniques protocol General details of cell culturing and sub-culturing The following is a general guideline for culturing of cell lines. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility. Contents Preparing an aseptic environment Preparation of cell growth medium Creating the correct cell culture environment Checking cells Sub-culturing Adherent subculture protocol Sub-culturing loosely attached cell lines requiring cell scraping for sub-culture Sub-culturing attached cell lines requiring trypsin Sub-culturing of suspension cell lines Changing media Passage number ​1) Preparing an aseptic environment Hood regulations (a) Close hood sash to proper position to maintain laminar air flow (b) Avoid cluttering Autoclaving (a) Pipette tips (or can be purchases pre-autoclaved, DNAse/RNAse free) (b) Glass 9” Pasteur pipettes (c) 70% ethanol (Be sure to spray all surface areas) ​All media, supplement, and reagents must be sterile to prevent microbial growth in the cell culture. Some reagents and supplements will require filter sterilization if they are not provided sterile. Watch our aseptic technique video protocol​ for detailed guidelines on avoiding contamination. 2) Preparation of cell growth medium Before starting work check the information given with the cell line to identify what media type, additives, and recommendations should be used. Most cell lines can be grown using DMEM culture media or RPMI culture media with 10% Foetal Bovine Serum (FBS), 2 mM glutamine and antibiotics can be added if required (see table below). Check which culture media and culture supplements the cell line you are using requires before starting cultures. Culture media and supplements should be sterile. Purchase sterile reagents when possible, only use unders aseptic conditions in a culture hood to ensure they remain sterile. ​​General example using DMEM media Media Measure DMEM - Remove 50 ml from 500 ml bottle, add other constituents. 450 ml 10% FBS 50 ml 2 mM glutamine 5 ml 100 U penicillin / 0.1 mg/ml streptomycin 5 ml 3) Creating the correct culturing environment Most cell lines will grow on culture flasks without the need for special matrixes etc. However, some cells, particularly primary cells, will require growth on special matrixes such as collagen to promote cell attachment, differentiation, or cell growth. We recommend reviewing the relevant literature for further information on the cells you are culturing. The following is an example for endothelial and epithelial cells: For human cells, coat flasks with 1% gelatin. Alternatively, for other cell types such as BAEC, flasks can be coated with 1% fibronectin. Prepare 10mL of coating solution composed of 1% gelatin or 1% fibronectin by diluting with distilled water, followed by filtration. This is efficient to coat about 5 flasks. Pipette coating solution into flask. Rock back and forth to evenly distribute the bottom of the flask. Let sit in an incubator for 15-30 minutes. Aspirate coating solution and wash with sterile dH2O before seeing cells. 4) Checking cells Cells should be checked microscopically daily to monitor health, grow rates and confluency (% surface area covered with cell monolayer). Adherent cells should be mainly attached to the bottom of the flask, show an adherent morphology (cell line dependant) and refract light around their membrane (refer to Abcam cell line data sheet images). Suspension cells should show a circular morphology and refract light around their membrane. Some suspension cells may clump (dissociation reagents such as Pluronic PF68 could be added to promote clump removal). Media that includes phenol red should be pink/orange in color (media color may change depending on CO2 environment). For imaging application media without phenol red can be used and will avoid interference with imaging acquisition. A pale yellow colour of media would indicate a acidity and decrease of pH which is often associated with contamination or unhealthy cells. Discard cells if: They are detaching in large numbers (attached lines) and/or look shrivelled and grainy/dark in color. They are in quiescence (do not appear to be growing at all). 5) Sub-culturing Also referred to as cell splitting and cell passaging. Split ratios or seeding densities can be used to ensure cells are ready for an experiment on a particular day or maintain cell cultures for future use or as a backup. Suspension cell lines are seeded based on volume so seeding densities will be calculated as cells/mL, whereas adherent cell lines are seeded based on flask surface area so will be calculated as cells/cm2. Cell lines often require specific seeding densities so always check the guidelines for the cell line in use. Slow growing cells may not grow if a high split ratio is used. Fast growing cells may require a high split ratio to make sure they do not overgrow. Adherent cell lines can be split using cell line specific split ratios or seeding densities (cells/cm2): 1:2 split should be 70-80% confluent and ready for an experiment in 1 to 2 days 1:5 split should be 70-80% confluent and ready for an experiment in 2 to 4 days 1:10 split should be 70-80% confluent and ready for sub-culturing or plating in 4 to 6 days. Split ratios are based on flask surface area, e.g.: 1 x 25 cm2 flask Split 1:3 would yield 3 x 25 cm2 flasks or 1 x 75 cm2 Suspension cell lines should be maintained using cell line specific seeding densities (cells/mL): 2e5 should be ready for an experiment in 3-4 days 1e6 should be ready for an experiment in 1-2 days If cells are to be left unattended for longer periods (i.e. bank holiday weekends) it is recommended to use a lower than normal seeding density/split ratio. ​ 6) Adherent subculture protocol (using dissociation reagent) When the cells are approximately 80% confluent (80% of the flask surface is covered by cell monolayer), cells should still be in their log phase of growth and will require subculturing. It is not recommended to allow cells to become over confluent as this may negatively affect gene expression and cell viability. ​ Remove cell culture media and dissociation reagent from the fridge and place in a 37oC incubator and allow to come to temperate 37oC. - Do not leave media in the incubator for longer than is necessary as the media components will degrade over time. ​ Switch on and perform a basic clean for your biological safety cabinet. - Spray all media bottles, pipettes and centrifuge tubes with ethanol before placing in the biological safety cabinet. Under the biological safety cabinet, remove the conditioned media and gently wash the cell monolayer with room temperature DPBS. - Carefully add DPBS to side of flask so not to forcefully dislodge adherent cells. Remove the DPBS using a sterile serological pipette and add pre-warmed dissociation reagent (Trypsin-EDTA) to the flask and place in an incubator for ~2 mins (dissociation times can vary between cell lines). Check flask frequently to ensure all cells have dissociated from flask surface. - Not all cells will require trypsinization, and to some cells it can be toxic. Trypsin can also induce temporary internalization of some membrane proteins, which should be taken into consideration when planning experiments. Other methods such as gentle cell scraping or using a very mild dissociation reagent (Versene) can often be used as a substitute in these circumstances. When all cells are detached, neutralise the dissociation reagent with serum containing growth medium appropriate to the cell line in culture. Transfer cell suspension to a centrifuge tube. Using sterile media, wash flask and transfer to centrifuge tube, ensuring all cells have been harvested from flask. Centrifuge the cell suspension for 5min @ 1000rpm, room temperature. Discard the supernatant and gently flick the cell pellet (to break up pellet), then resuspend cells in sterile media to a suitable volume for counting. Consult Abcam counting cell using a hemocytometer protocol. Based on count and viability data, seed cell suspension for an appropriate flask and density, e.g. T175, 30mL at 2e4 cells/cm2. - Label culture flask with all necessary info e.g. Cell Line, passage number, etc. Immediately incubate the newly seeded cultures in a 37oC/5% CO2 air humidified incubator. 7) Sub-culturing loosely attached cell lines requiring cell scraping for sub-culture When ready, carefully pour off media from flask of the required cells into waste pot (containing approximately 100 ml of 10% sodium hypochlorite) taking care not to increase contamination risk with any drips. Replace this immediately by carefully pouring an equal volume of pre-warmed fresh culture media into the flask. Using cell scraper, gently scrape the cells off the bottom of the flask into the media. Check all the cells have come off by inspecting the base of the flask before moving on. Take out required amount of cell suspension for required split ratio using a serological pipette. e.g. for 1:2 split from 100 ml take 50 ml into a new flask 1:5 split from 100 ml take 20 ml into a new flask 1:10 split from 100 ml take 10 ml into a new flask Top the new flasks up to required volume (taking into account split ratio) with pre-warmed fresh culture media eg. in 25 cm2 flask approx 5-10 ml 75 cm2 flask approx 10-30 ml 175 cm2 flask approx 40-150 ml 8) Sub-culturing attached cell lines requiring trypsin Note – not all cells will require trypsinization, and to some cells it can be toxic. It can also induce temporary internalization of some membrane proteins, which should be taken into consideration when planning experiments. Other methods such as gentle cell scraping, or using very mild detergent can often be used as a substitute in these circumstances. When ready, carefully pour off media from flask of the required cells into waste pot (containing approximately 100 ml 10% sodium hypochlorite) taking care not to increase contamination risk with any drips. Using aseptic technique, pour/pipette enough sterile PBS into the flask to give cells a wash and get rid of any FBS in the residual culture media. Tip flask gently a few times to rinse the cells and carefully pour/pipette the PBS back out into waste pot. This may be repeated another one or two times if necessary (some cell lines take a long time to trypsinize and these will need more washes to get rid of any residual FBS to help trypsinization) Using pipette, add enough trypsin EDTA to cover the cells at the bottom of the flask. e.g. in 25 cm2 flask approx 1 ml 75 cm2 flask approx 5 ml 175 cm2 flask approx 10 ml Roll flask gently to ensure trypsin contact with all cells. Place flask in 37°C incubator. Different cell lines require different trypsinization times. To avoid over-trypsinization which can severely damage the cells, it is essential to check them every few minutes. As soon as cells have detached (the flask may require a few gentle taps) add some culture media to the flask (the FBS in this will inactivate the trypsin) Using this cell suspension, pipette required volume of cells into new flasks at required split ratio. These flasks should then be topped up with culture media to required volume e.g. in 25 cm2 flask approx 5-10 ml 75 cm2 flask approx 10-30 ml 175 cm2 flask approx 40-150 ml Leave cells overnight to recover and settle. Change media to get rid of any residual trypsin. 9) ​Sub-culturing of suspension cell lines​ Check guidelines for the cell line for recommended split ratio or subculturing cell densities. Take out required amount of cell suspension from the flask using pipette and place into new flask. ​e.g.For 1:2 split from 100 ml of cell suspension take out 50 ml ​For 1:5 split from 100 ml of cell suspension take out 20 ml Add required amount of pre-warmed cell culture media to fresh flask. e.g. For 1:2 split from 100 ml add 50 mls fresh media to 50 ml cell suspension ​For 1:5 split from 100 ml add 80mls fresh media to 20 ml cell suspension 10) Changing media If cells have been growing well for a few days but are not yet confluent, then they will require a media changing to replenish nutrients and keep correct pH. Cells produce positive growth promoting factors which are secreted into their media so it can be beneficial to perform a half media change replenish nutrients provided by the media and also maintain these positive growth factors. To change media, warm up culture media at 37°C using a water bath or incubator for at least 30 min. Aspirate old media from the flask and replace the media with the necessary volume of fresh pre-warmed culture media and return to incubator. ​ 11) Passage number The passage number is the number of sub-cultures the cells have gone through. Passage number should be recorded and not get too high. This is to prevent use of cells undergoing genetic drift and other variations. 

Abstract Undergraduate students learn about mammalian cell culture applications in introductory biology courses. However, laboratory modules are rarely designed to provide hands-on experience with mammalian cells or teach cell culture techniques, such as trypsinization and cell counting. Students are more likely to learn about cell culture using bacteria or yeast, as they are typically easier to grow, culture, and manipulate given the equipment, tools, and environment of most undergraduate biology laboratories. In contrast, the utilization of mammalian cells requires a dedicated biological safety cabinet and rigorous antiseptic techniques. For this reason, we have devised a laboratory module and method herein that familiarizes students with common cell culture procedures, without the use of a sterile hood or large cell culture facility. Students design and perform a time-efficient inquiry-based cell viability experiment using HeLa cells and tools that are readily available in an undergraduate biology laboratory. Students will become familiar with common techniques such as trypsinizing cells, cell counting with a hemocytometer, performing serial dilutions, and determining cell viability using trypan blue dye. Additionally, students will work with graphing software to analyze their data and think critically about the mechanism of death on a cellular level. Two different adaptations of this inquiry-based lab are presented—one for non-biology majors and one for biology majors. Overall, these laboratories aim to expose students to mammalian cell culture and basic techniques and help them to conceptualize their application in scientific research. Go to: INTRODUCTION Background One of the most important model systems for researchers in numerous fields of biology and medicine is the culture of mammalian cells or, more specifically, the growth and dispersal of cells derived from animal tissues, with an appropriate surface, proper nutrients, and a suitable environment. While the culture of cells and tissues has been in practice since the late 1800s, it was not until the early 1950s, with the remarkable growth of cells biopsied from the cervical cancer of Mrs. Henrietta Lacks, that the practice began to yield significant discoveries. The successful culture of Mrs. Henrietta Lacks’s cells, now called HeLa cells, effectively revolutionized biological and medical research, enabling countless cellular, molecular, and therapeutic breakthroughs, including the discovery of the first effective polio vaccine (1,2). Today, the HeLa cell line has yielded more than 60,000 publications and has been instrumental in several Nobel prize-winning discoveries (3,4). Moreover, the publication of the book The Immortal Life of Henrietta Lacks in 2011 generated popular interest in human cell culture and techniques, even prompting many high schools to assign the book as required reading (5). Despite the now widespread use of mammalian cell culture as a model for biological research, many undergraduate biology students are not exposed to the technique until upper level classes or even graduate level research. The reason for the lack of mammalian cell culture-based laboratory modules is the large and expensive equipment requirements and rigorous sterile-technique training that are not well suited to a large classroom environment. It is for this reason that we have devised a straightforward approach to teaching common mammalian cell culture techniques to undergraduate students in an introductory biology class. The approach can be adapted to both biology major and nonmajor classrooms with a few simple changes. In this paper, we describe how HeLa cells were used as a mammalian model to teach undergraduates about cell culture techniques including cell manipulation, trypsinizing cells, counting, and determining cell viability. Three different adaptations of this inquiry-based lab are presented—one for non-biology majors, one for biology majors, and one for honors biology majors. Overall, this approach allows biology students to acquire and develop skills useful to them in subsequent experiences in cell culture laboratories while gaining an introductory understanding of how scientists use mammalian cells in research. Furthermore, we find that this laboratory stimulates reflection and discussions related to biomedical ethics and consent in scientific research. Intended audience/Prerequisite knowledge This laboratory exercise is intended for undergraduate students in introductory biology courses, with details provided to tailor the experience to students in a general biology education class (nonmajors) or biology majors. This laboratory explores concepts related to mammalian cell culture, the effects of different compounds on cell viability, and cell manipulation. For students in the general biology education class, this also dovetails into an introduction to the history of cell culture and the contribution of Mrs. Henrietta Lacks, as well as the ethical implications of this research. Biology major students will be expected to select and design their experimental conditions, set up serial dilutions, and employ appropriate controls in order to study mammalian cell viability. Therefore, prior to the beginning of the laboratory period, students should be familiar with terms such as HeLa cells, trypsin, trypan blue, and cell viability and have been introduced to the concept of experimental design. Prior pipetting practice, sterile technique, and acquaintance with a compound microscope are encouraged. Learning time General education biology (nonmajors) Students were given a reading assignment by Rodríguez-Hernández et al. to complete before the lab period, introducing the historical background of cell culture as well as its application in research (1). To complete the laboratory in its entirety takes one full lab period of approximately two hours, since the cell treatment option has been limited to pre-selected temperatures. It is helpful if students are already familiar with the compound microscope; however this is not a requirement. Incubation times can be used to provide students with more information on the history of HeLa cells and to discuss the ethics surrounding their initial collection and use in research today. Students are expected to collect all of their data within the allotted two hours, but they will conduct their analysis and generate figures following the lab period. Biology majors To complete the laboratory in its entirety will take one lab period of approximately three hours, provided that students have already been introduced to dilution calculations and are comfortable with the use of disposable bulb pipettes, micropipettes, and compound microscopes. Prior to the lab, students should be given a list of potential compounds and treatments from which to choose so that they may study and research the cellular mechanism by which their condition causes cell death. A list of potential treatments and suggested ranges is listed in Table 1. This inquiry-based learning lab will help the students develop a hypothesis related to cell viability and the dosage of their compound of interest. Students are expected to collect all of their data within the three hours, but they will generate their figures, methods, and results sections outside of the given lab time. TABLE 1 HeLa cell treatment options and suggested ranges. Treatment Low Medium High Temperature 4ºC 37ºC 42ºC pH 2 7 12 NaCl 0 M 0.1 Ma 0.5 M KCl 0 M 0.1 Ma 0.5 M NaF 0 M 0.1 Ma 0.5 M Ethanol 0% 25% 50% Treatments are expressed in final concentrations. All solutions were prepared in 1 × PBS. aConcentration based on the approximate amount of NaCl present in PBS. PBS = phosphate-buffered saline. Learning objectives Upon completion of this lab, students will be able to: Perform the basics of cell culture technique, including trypsinizing cells and cell counting, and determine cell viability using trypan blue exclusion. Utilize dilution math and graphs to analyze cell viability data. Create a scientific figure that conveys cell viability data. Go to: PROCEDURE Materials A graphic outline of the procedure is shown in Figure 1. Special materials include the following: An external file that holds a picture, illustration, etc. Object name is jmbe-18-38f1.jpg FIGURE 1 A schematic outlining the experimental procedure to count cells. PBS = phosphate-buffered saline; FBS = fetal bovine serum. HeLa cells (ATCC, CCL-2) minimum essential medium (GIBCO DMEM High Glucose, Pyruvate) 0.05% (w/v) trypsin-EDTA solution (GIBCO, 25-300-054) fetal bovine serum (GIBCO, 26-140-087) phosphate buffered saline (GIBCO, 10-010-023) trypan blue solution, 0.4% (GIBCO, 15-250-061) traditional hemocytometer or a Glasstic Slide 10 with Counting Grids (Kova International, 87144) – one per pair of students Standard materials for the laboratory preparation include tissue culture-treated T-75 flask (to expand the cells) serological pipettes centrifuge tubes tissue culture-treated culture dishes (Fisher Scientific, 12-565-94) bulb pipettes P20 pipettes and tips microcentrifuge tubes 15 mL centrifuge tubes Faculty or laboratory personnel should conduct the initial HeLa cell growth, stock freezing, and dish preparation in a cell culture hood; however, all experiments described herein can be conducted in a standard undergraduate biology laboratory. Cell counts are collected using a compound light microscope fitted with 10×, 20×, and 40× objectives. Materials do not have to be sterile as cells will be discarded once the short experiments have been completed. A water bath to warm reagents to 37ºC (ideally proximal to the student work area) is recommended, but not required. Detailed instructions and ordering information can be found in the student/instructor guides (Appendices 1, 3, and 5). Student instructions HeLa cell washing and dissociation Students were provided with a petri dish of HeLa cells at 80 to 90% confluence. Culture media was removed with a disposable bulb pipette and cells were rinsed by adding 5 mL of 1 × PBS and gently rocking the plate. After 1 min, the PBS was removed using a bulb pipette, and 1 mL of a 0.05% trypsin-EDTA solution was added. The plate was gently rocked back and forth to ensure that the trypsin entirely covered the cells. After 10 minutes of periodic rocking, 1 mL of 1% FBS (in 1×PBS) was added to the plate and mixed gently with the cell/trypsin mixture in order to stop the enzymatic digestion by trypsin. Finally, the 2 mL mixture of cells and media was transferred from the culture dish to a sterile 15-mL conical centrifuge tube and gently mixed with an additional 8 mL of pre-warmed 1 × PBS. HeLa cell counting Once the cells were dispersed, 20 μL of cell suspension was transferred to a microcentrifuge tube; 20 μL of trypan blue was then added and mixed gently with a pipette; 20 μL of cell/trypan blue suspension was then loaded into a cell counting chamber and placed under the microscope for counting. HeLa cell viability—general biology education To investigate HeLa cell viability, nonmajor students were directed to split an equal volume of their cells into nine microfuge tubes, and incubate three replicates each at temperatures of 4°C, 37°C, and 42°C, respectively. After 30 minutes, cells should be counted via the trypan blue method described above and data should be recorded for subsequent analysis. HeLa cell viability—biology majors Biology majors were instructed to pick a chemical or treatment from a list provided. Table 1 provides some examples of treatments and suggested ranges. For example, students can elect to treat their cells with various concentrations of ethanol ranging from 0 to 25%. To provide a more inquiry-based laboratory, students can also be given general instructions to determine their own treatments and/or determine three diverse treatment conditions. Based on cell concentrations determined in the previous step, biology majors should then be able to calculate how to dilute their cells to a concentration of approximately 4 × 104 cells/mL using PBS as the diluent. To run three replicates each for three treatments and a negative control (untreated sample), 500 μL of the 4 × 104 cells/mL cell suspension should be pipetted into 12 microcentrifuge tubes. After treating with the appropriate chemical or condition, cells should be counted via the trypan blue method described above. For students selecting a drug or chemical, the treatment should increase the total volume by no more than 500 μL to avoid low cell counts. Also, it is important that the final volume be consistent for all treatments. For example, if the student adds 100 μL of compound solution at the highest concentration to the cell suspension, the control should receive 100 μL of 1 × PBS and other experimental treatments should have a total of 100 μL added (compound solution + 1 × PBS). Faculty instructions Faculty should be familiar with techniques involving basic cell culture and manipulation, including passaging and freezing of cells (to maintain HeLa cell stocks). For the chemical or drug selection, we allowed biology majors to do the math to determine their treatment concentration. However, to simplify the exercise, we provided stock solutions (for example 50% ethanol in PBS). Faculty should familiarize themselves with basic cell counting techniques, including the equations needed to determine cell concentrations using hemocytometers or cell counting chambers, as they can differ significantly. Preferred software for graphing is Microsoft Excel; however, Google Sheets can also be used to perform the analysis. To keep costs to a minimum, each lab student pair or group was only given one plate of cells and the minimum reagents needed to perform these experiments. A detailed preparation guide for instructors, including ordering information for a disposable and easy-to-use hemocytometer, is provided in Appendix 5. Suggestions for determining student learning Both biology nonmajors and majors were instructed to generate a scientific figure and a write-up detailing their initial hypothesis and indicating whether their data supported their hypothesis and why they thought this was the case. If no differences were found between treatments, students were asked to explain what might have occurred to produce these results. In addition, biology majors were required to include methods and results sections to accompany their scientific figure, to include error bars, and to perform a Student’s t-test to determine whether their results were significant. Sample data—general biology education In this laboratory, we determine the effects of diverse temperatures on the viability of HeLa cells. A sample of cells was taken from each treatment, mixed with an equal volume of trypan blue and pipetted into a hemocytometer or cell counting slide. Using a compound microscope, students were able to differentiate the number of viable (excluded trypan blue) and nonviable (took up trypan blue) cells from each of the three replicates at three different treatment temperatures. For Glasstic Slide 10 with Counting Grids, cell viability was then determined using the following equation: Live cell concentration (cells/mL)=(# live cells# squares)×7,500×trypan blue dilution On average, students counted cells in a total of 18 squares out of a possible 81 to get a live cell concentration estimate. Figures 2A and 2B show representative student figures generated from the data collected in this experiment. Additional samples of student work can be found in Appendix 2. The rubric used by instructors to grade students’ work is provided in Table 2, and the average student scores on the lab deliverable are provided in Figure 4. An external file that holds a picture, illustration, etc. Object name is jmbe-18-38f2.jpg Open in a separate window FIGURE 2 Data from non-biology majors representing HeLa cell viability based on temperature. Student viability counts are shown from the incubation of HeLa cells at 4°C, 37°C, and 42°C. A) Representative data from a student pair that graphed average cell counts. Standard deviation is indicated as error bars where n=3 for all treatments. B) Representative data from a student pair that graphed individual cell counts for each treatment. TABLE 2 Rubric for assessment of student deliverables. Criteria Points Correct type of graph 1 Axes are labeled appropriately (including units) 1 Axis units and ranges are appropriate 1 Reasonable data values 1 Appropriate statistics in graph (e.g., error bars or p values) 2 Data categories are clear (in legend or key) 1 Figure legend is clear and well written 2 Descriptive figure title in figure legend 1 TOTAL POSSIBLE SCORE 10 An external file that holds a picture, illustration, etc. Object name is jmbe-18-38f4.jpg Open in a separate window FIGURE 4 Student scores on lab deliverables suggest learning objectives were met. Student lab graphs were graded by instructors using the assessment rubric described above (Table 2). The values shown correspond to students taught in the fall of 2016 (n > 100). A) Student average scores for both Biology majors and nonmajors. Standard deviation is indicated as error bars. B) Student scores as a percentage of students attaining a failing, passing, or high passing score. Sample data—biology majors In the modified version of the lab for biology majors, we examined the effect of various treatments on the viability of HeLa cells. As outlined above, cell viability was determined using trypan and a designated cell counting chamber. Figures 3A and 3B show representative student figures generated from cells treated with diverse concentrations of ethanol and NaCl, respectively. The rubric used by instructors to grade students’ work is provided in Table 2 and the average student scores on the lab deliverable are provided in Figure 4. An external file that holds a picture, illustration, etc. Object name is jmbe-18-38f3.jpg Open in a separate window FIGURE 3 Data from biology majors representing HeLa cell viability based on diverse treatments. A) Student viability data from the incubation of HeLa cells with ethanol concentrations ranging from 0 to 25%. Standard deviation is indicated as error bars where n=3 for all treatments. B) Student viability data from the incubation of HeLa cells with NaCl concentrations ranging from 0 to 0.5 M. Standard deviation is indicated as error bars where n=2 for all treatments. Safety issues Before the first day of lab, students were required to complete a reading and watch a series of third-party videos on lab safety. After completing these assignments, compliant with the ASM Guidelines for Biosafety in Teaching Laboratories (specifically those for working with BSL-2 materials like HeLa cells), students were required to sign a lab safety agreement confirming that they would abide by these guidelines (6). For instructors to be sure students understood the key points of lab safety, students completed a quiz on lab safety and were required to obtain a passing grade on this assessment before continuing to engage in lab activities. On the first day of lab, lab instructors reviewed the highlights of lab safety and students were given the opportunity to ask additional questions or share any concerns. In the weeks leading to the experiments described in this manuscript involving HeLa cells (BSL-2), students were required to demonstrate they had the skills required to work with BSL-2 materials using a BSL-1 organism, the budding yeast Saccharomyces cerevisiae. In these experiments, students tested the ability of yeast to survive UV treatment or to grow in different carbon sources. In this way, students demonstrated that they could safely and appropriately handle BSL-2 organisms such as HeLa cells. Students working with HeLa cells (BSL-2) and chemical reagents must wear lab coats, gloves, closed-toed shoes, and protective eyewear during the experiment. Trypan blue stain can cause irritation to the eyes, skin, and respiratory system if protective wear is not utilized. Disposal of BSL-2 materials and reagents should be contained to appropriate biohazard containers within the laboratory. Waste should be autoclaved or bleached before disposal. In accordance with ASM guidelines, students should maintain a clean workspace (ethanol spray bottle should be provided), wear gloves, lab coats, and safety goggles, as well as thoroughly wash their hands before entering and exiting each laboratory session (6). Go to: DISCUSSION Field testing This activity was implemented in fall 2015, spring 2016, summer 2016, and fall 2016 in general biology courses at High Point University. A total of 595 students experienced this inquiry-based laboratory activity. Evidence of student learning—general education biology A student-generated cell viability graph and analysis are included in Appendix 2 as an example of student learning. It is clear from these data that students were able to successfully manipulate, treat, count, and assess cell viability. Further, students were able to thoughtfully consider the data and relate it to their own knowledge of human cells by highlighting the increased viability observed at 37°C. In their post-lab reflection surveys, students indicated that they learned about the importance of precision and accuracy in scientific work in the lab, as well as the technical skills acquired including trypsinization and cell manipulation. Students also indicated that they enjoyed how “hands on and active it was” and that they were “actually using human cells” in the lab. Students tended to dislike the length of the lab, which took a full two hours of active engagement. A detailed summary of student reflections is included in the preparation guide for instructors provided in Appendix 5. Evidence of student learning—biology majors A cell viability chart and analysis produced by a biology major is included in Appendix 4. The figure, legends, and axes demonstrate that students were able to successfully design and implement their experiment, as well as present their data in a way that facilitates meaningful analysis. The students were also capable of including the correct control (0% ethanol) to determine the effect of their treatment by comparison. It is clear from this data that students were able to convey their findings in a way that allowed them to then draw conclusions based on the given treatment. In their post-lab reflection surveys, students indicated how much they enjoyed being able to select their individual treatments and “design an experiment,” as well as the ability to work with and visualize human cells. In contrast to the nonmajors students, more biology majors tended to dislike counting the cells, a process they found monotonous, and disliked “waiting for the cells to finish incubating.” Evidence of student learning—lab deliverables To demonstrate what they had learned, biology majors and nonmajors were required to construct a scientific figure with their data to convey their findings. The rubric used by instructors to grade students’ work is provided in Table 2, and the average student scores on the lab deliverable are provided in Figure 4. The items assessed on student deliverables were designed to help the instructors determine whether the students had been able to meet the first three learning objectives of the laboratory module (Table 2). A passing student average score suggests the majority of students met these first three learning objectives (Fig. 4). Possible modifications The purpose of these experiments is to introduce students to the basic principles of cell culture, determine the effects of various treatments on cell viability, and analyze the data using a data analysis software package. As mentioned, a possible modification for a nonmajors biology course could be restricting treatment types to temperature only. This would avoid much of the math associated with dosing and allow for the lab to be completed in a shorter period (see Appendix 3 for non-biology major student lab handout/guide). Another extension, if equipment and resources are available, is for students to engage in a longer multi-week project, which could be suitable for an honors track. For example, students could study the effects of a nicotine treatment on HeLa cells (100 nM up to 100 μM), or a similar substance, for at least 12 hours and up to two days, thereby studying the effects of both concentration and time. In addition to assessing cell survival rates, students could also isolate RNA, and utilize RT-PCR to examine alterations in the expression levels of candidate proliferation genes. Students could be asked not only to test a hypothesis generated based on the effects of low to moderate levels of nicotine exposure and the influence of incubation time on cell viability and the expression of candidate proliferation genes, but to determine whether HeLa cells express their gene of interest and to examine whether their candidate genes change in expression levels as a result of nicotine exposure. Go to: SUPPLEMENTAL MATERIALS Appendix 1: Student handout – non-biology majors, Appendix 2: Sample student analysis – non-biology majors, Appendix 3: Student handout – biology majors, Appendix 4: Sample student analysis – biology majors, Appendix 5: Instructor preparation guide Click here for additional data file.(1.9M, pdf) Go to: ACKNOWLEDGMENTS This experiment was utilized in fall 2015, spring 2016, summer 2016, and fall 2016 in general biology courses at High Point University, and the authors appreciate the efforts of the students. Funding was supplied by the Department of Biology at High Point University and a Roberta Williams Laboratory Teaching Initiative Grant from the Association for Biology Laboratory Education to V.A. Segarra. The authors declare that there are no conflicts of interest. Go to: Footnotes †Supplemental materials available at http://asmscience.org/jmbe Go to: REFERENCES 1. Rodríguez-Hernández CO, Torres-Garcia SE, Olvera-Sandoval C, Ramirez-Castillo FY, Muro AL, Avelar-Gonzalez FJ. Cell culture: history, development and prospects. Int J Curr Res Acad Rev. 2014;2:188–200. [Google Scholar] 2. del Carpio A. The good, the bad, and the HeLa. Berkley Sci Rev. 2014. Spring. http://berkeleysciencereview.com/article/good-bad-hela/ 3. Masters JR. HeLa cells 50 years on: the good, the bad and the ugly. Nat Rev Cancer. 2002;2:315–319. doi: 10.1038/nrc775. [PubMed] [CrossRef] [Google Scholar] 4. Schwarz E, Freese UK, Gissmann L, Mayer W, Roggenbuck B, Stremlau A, zur Hausen H. Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature. 1985;314:111–114. doi: 10.1038/314111a0. [PubMed] [CrossRef] [Google Scholar] 5. Skloot R. The immortal life of Henrietta Lacks. Random House; New York, NY: 2010. [Google Scholar] 6. Emmert E. Biosafety guidelines for handling microorganisms in the teaching laboratory: development and rationale. J Microbiol Biol Educ. 2013;14(1):78–83. doi: 10.1128/jmbe.v14i1.531. [PMC free article] [PubMed] [CrossRef] [Google Scholar] Articles from Journal of Microbiology & Biology Education are provided here courtesy of American Society for Microbiology (ASM)

1. Pursue post-graduation in biotechnology DUO-India Fellowship Opting for post-graduation in biotechnology can lead to much advancement in your career as this field is acknowledged by having considerable knowledge. Post-graduation will not only increase your knowledge about this field but would increase the job opportunities in both the private and government sectors. You can apply for exams like GATE and CSIR NET through which you can pursue your higher education in a well-recognized university in India as well in foreign. You can also go for some prestigious institutions like IIT, JNU and AIMS for your master’s degree. 2. Work under a scientist PAU Biotech You can work as an assistant researcher under a scientist doing his project in the related field if you are discontinuing your studies or planning to take a year off. This will give you the experience to work in laboratories and will also improve your lab skills. You won’t be paid much for this job, but an experience certificate will help you in the long term of your career. If you ‘re a hard worker, you may also be promoted where you can conduct your experiments or research work. 3. Apply for a job in the private sector There is no end to biotechnology industries and sectors. These private jobs seek for biotechnology experts that are way too good for their company. The fresh biotech graduates can apply for a job in these sectors and can earn very well. There are ample industries that hire freshers, some of the major recruiters include chemical and pharmaceutical industries, bioprocessing industries, cosmetic industries, companies associated in manufacturing and developing agricultural and biological products, some healthcare products producing companies and so on. So if you are a company person, you can opt-out for these options once you are graduated. 4. Work as a Laboratory Technician/Assistant By so far, this can be one of the most convenient jobs you can settle on right after your graduation.There are some private as well as government universities and laboratories that look out for lab managers and technical assistants who can easily handle laboratory equipment and can perform laboratory tests or techniques.While choosing this as a career option, you can also indulge yourself to do some lab work, working with specialized lab equipment and machines and much more. The pay scale for this job is not expected much, but you can gain an ample amount of practical knowledge. 5. Become an entrepreneur Becoming an entrepreneur is one of the ambitions for young minds. Starting something of your own and taking it to heights is something one of the best career options in biotechnology. Also being a biotechnology student you can do wonders in these fields. Like setting up your own company to design various drugs, developing RNA Therapies, storing of cultures and cells for laboratories. So if you want to be your boss in life and work according to yourself and not under a fixed schedule, being an entrepreneur will be an ideal job for you.You can get inspired by various startups that have been established in the work of research. If you have original ideas, you can also get funded by the Indian government and can start your Biotech revolution. 6. Apply for the job of Sales in a Biopharma company Being a Biotech graduate, doors are always open for the ones who wish to make their career in biotech companies. There are various posts and opportunities provided for job seekers just after their graduation. Opting for non-scientific jobs like becoming a sales agent or medical representative will indulge your career in the field of biotechnology. You can get recruited in the best companies like Biocon, Panacea Biotec Limited, Dr. Reddy’s Laboratories Limited, Novozymes and much more. Being a sales agent you “ll get to deal with doctors, researchers, techs, students or pharmaceutical scientists. 7. Apply in the government sector There are various opportunities for the graduates to make their career in government sectors as well. In India, Biotech developments are monitored under the Department of Biotechnology which is under the control of the Ministry of Science and Technology. Vacancies for various posts are always expected. In the government sector, you can also work in different research institutions or public health care centres. 8. Get a job in Research and Development (R&D) If you are planning to take a break for a year before going for postgraduation, you can get placed for a job in the Research and Development in India. Working for R&D would be one of the best career options in biotechnology for the ones seeking further with their MSc or Ph.D. You can get to know a lot about how research work is carried out. Also, if you are much dedicated to your work you will be regularly promoted to high positions which will also increase your income. 9. Get a job in Intellectual Property Research and Patenting We all might have heard about copyright or patenting something which is your discovery. By getting a job in this sector you “ll work with the agencies that manage to a patent of a discovery. You can start with the prominent patent firms of India. In this filed you”ll get to know about different laws which can help you in legal matters.Also, if you wish to go for a law degree, you can become a Biotech patent lawyer. 10. Be a teacher in an Organization/university By getting graduated in Biotechnology you can also choose teaching as a career option. Being only a graduate student the only option is to teach in schools. But if you want to be a lecturer in universities you must have a Master’s degree along with the clearance of the exam NET (National Eligibility Test). For the assistant professor position Ph.D. is mandatory. So if you are passionate about teaching, you can settle as a teacher or a lecturer in your future. So here we end our list of best career options in biotechnology. Overall you have plenty of options, just keep in mind that in order to get the best, you need to be best. So study well, work hard and work on your skills as well as personality. Surely you will make a good career in biotechnology.

BIOTECH Project Activities The BIOTECH Project has worked with over 100,000 students across Arizona in the past six years. Hundreds of teachers have brought engaging hands-on biotechnology activities to their classroom through professional development workshops, classroom visits and material and equipment loans. Due to budget cuts, materials cost is now associated with the activities. Download the price list, however email Nadja(link sends e-mail) to get the most current prices. To request Biotech resources please submit a resource request form here or email Nadja Anderson at nadja@bio5.org(link sends e-mail). High School Activities ****COVID19 Remote Instruction--With all instruction being online we are working to create online resources for teachers to use. These will include instruction about SARS CoV 2 and COVID19 as well as some of our activities below. COVID19 Teaching Resources--Resources include links to understand the virus, testing for the virus (both RT PCR and Immunological test), as well as activities around the virus. Penicillium Antibiotic Effect How were antibiotics discovered? How is the effect of an antibiotic different for different species of bacteria? This activity touches on the history of early antibiotics research as well as serves as an example of how observation leads to discovery. We have optimized a set of experiments to fit the high school classroom working with an antibiotic producing fungus and species of bacteria to emulate early observations of antibiotic effect on bacteria. Students normalize cultures of Penicillium fungi (green bread mold) as well as bacterial species Staphylococcus epidermidis, Microcuccus luteus, and Enterobacter aerogenes using spectrophotometry before co-culturing the fungus with the bacteria to witness the antibiotic effect. Students then measure the results of co-cultivation by quantifying the optical density of the bacteria at the end of one week of experimentation. [5 days] [Student Guide] [Teacher Guide] [Penicillium Antibiotic Effect Page] [Materials Guide] [Material MSDS List] Kiwi DNA Extraction How do you purify DNA from cells? Students extract DNA from kiwifruit to learn about the chemical and physical properties of DNA. This activity provides a first-hand understanding of how DNA can be isolated for further analysis, such as DNA fingerprinting. Students also reinforce their understanding of cell structure and biological macromolecules. We use a kiwifruit protocol because it uses commonplace materials and requires little equipment. [45 minutes] [Student Guide][Teacher Guide] [Materials Guide] [Materials MSDS List] Agarose Gel Electrophoresis with Dyes What is electrophoresis? Students use agarose gel electrophoresis to determine the composition of different biological materials. This activity helps students learn how molecules can be separated and identified by electrophoresis. [50-60 minutes] [Student Guide][Teacher Guide] [Materials Guide] [Materials MSDS List] DNA Fingerprinting How is DNA evidence prepared and analyzed in a crime case? Students perform agarose gel electrophoresis to analyze DNA samples from a mock crime scene. Based on DNA fingerprinting profiles that are simulated to represent the three suspects, and DNA from the crime scene, students determine which suspect likely committed the crime. This activity helps students understand how DNA variation in individuals can be analyzed in practical applications such as genetic testing and forensics. [120 minutes—One block period + part of one normal period] Some Examples: Cat Food Caper - [Student Guide] [Teacher Guide] [Materials Guide] [Materials MSDS List] Bubble Gum Mystery - [Student Guide] [Teacher Guide] [Materials Guide] [Materials MSDS List] Lipstick Mystery - [Student Guide] [Teacher Guide] [Materials Guide] [Materials MSDS List] Who took a bite out of the Principal’s cookie? - [Student Guide] [Teacher Guide] [Materials Guide] [Materials MSDS List] Abuela Project - [Student Guide] [Teacher Guide] [Materials Guide] [Materials MSDS List] Whale Paternity - [Student Guide] [Teacher Guide] [Materials Guide] [Materials MSDS List] Huntington’s Disease Clinical Investigation and DNA Electrophoresis This activity will allow students to evaluate two patients with possible neurological symptoms. Students will come up with possible diagnosis and determine how to test for these diagnoses. The activity is completed with an electrophoresis to test for Huntington’s disease in the patients. [One 50 min class period for introduction, 1-3 class periods for research/diagnosis & tests reporting, 50 min gel electrophoresis, and partial class period follow-up and final analysis] [Student Guide] [Teacher Guide] [Materials Guide] [Diagnosis Worksheet] [Materials MSDS List] Sickle Cell Anemia A patient and his wife come in to see you with a concern. The patient has a history of sickle cell disease in his family, but neither of his parents have exhibited any symptoms. The wife is an immigrant from rural tropical Africa and has no idea if her family has any history of sickle cell disease. However the area she is from has a high incidence of sickle cell anemia in the population. The couple has 2 children, ages 4, 8 and would like to have another. Their kids don’t know about the history of the disease. The couple has come to you for advice on whether or not to have another child, and what to tell their children about the family medical history. [One 50 min class period for introduction, 50 min gel electrophoresis, and partial class period follow-up and final analysis] [Student Guide] [Materials Guide] [Teacher Guide] [Materials MSDS List] PTC--To Taste or Not to Taste Why do you think some students can taste the PTC and others can’t? Are you a taster or not? Students will test the DNA of Jillian and her family for the PTC gene and determine if their genetics correlates with the tasting data. The most common PTC gene mutation (resulting in the inability to taste PTC) in the US population is due to a deletion of part of the gene, which is easily tested for and visualized by DNA electrophoresis. Students will use this information to help them draw a family tree for Jillian. [Student Guide] [Materials Guide] [Materials MSDS List] Restriction Enzyme Analysis How is DNA analyzed and manipulated using restriction enzymes? Students digest bacteria phage lambda DNA with different restriction enzymes and analyze the resulting DNA profiles. Students compare the DNA fragments with the known restriction map of bacteria phage lambda. This activity demonstrates how DNA sequences can be mapped and characterized, such as in the Human Genome Project and how DNA is cut and arranged during genetic engineering. [50 minutes, overnight incubation, 90 minutes, plus 50 minutes] [Student Guide] [Teacher Guide] [Materials Guide] [Materials MSDS List] Bacterial Transformation-Mystery - Teaches Genotype to Phenotype Concepts What is genetic engineering, and how is this technique used? Students perform a genetic engineering experiment using bacterial transformation to introduce fluorescent genes into Escherichia coli (E. coli), to produce bacteria that fluoresce different colors or "glow". This activity helps students understand what genes do and how they can be manipulated by genetic engineering. This activity will confirm that different genes introduced by transformation will result in different visible characteristics. [Student Guide] [Teacher Guide] [Materials Guide] [Materials MSDS List] Bacterial Transformation-Regulation- Teaches Gene Regulation/Inducible Promoter Transform E. coli with green fluorescent protein gene and observe its regulation with an inducible promoter. This activity helps students to visualize regulation and relate this regulation to the lac operon system. Highly recommended for AP Biology. This activity can be combined with a PCR investigation to confirm that the GFP gene is present in the non-induced E. coli see PCR (A). [50 minutes, overnight incubation, and part of the next 50 min class] [Student Guide] [Teacher Guide] [Materials Guide] [Materials MSDS List] Bacterial Transformation with GFP combined with PCR for GFP This activity allows the gene regulation concept to be presented in a more inquiry fashion. Students will transform E. coli with green fluorescent protein gene and will observe the absence of glowing protein. They will hypothesis why the cells did not glow and use PCR (B) to test their hypothesis. After the PCR the students will learn the regulation of this gene, and will induce the promoter to express the product. This activity helps students to visualize regulation and relate this regulation to the lac operon system. Highly recommended for AP Biology and Biotechnology Course. [50 minutes, overnight incubation, and next 50 min class, then PCR (see below) then another one or two 50 min class(es),depending on how much is inquiry, plus a part of a class to complete the activity] [Student Guide] [Materials Guide] [Materials MSDS List] PCR of GFP (staining with Methylene Blue) In this lab investigation students will learn about a technique called polymerase chain reaction (PCR) that allows us to examine a very small piece of DNA. The piece of DNA that is replicated is called the Green Fluorescent Protein (GFP) gene. This gene codes for the GFP protein, a protein normally produced by jellyfish that is transformed into bacteria in a plasmid (pGLO). This activity lends itself to be conducted inquiry style. [Student Guide] [Materials Guide] [Materials MSDS List] Extra material: * Sequencing the PCR Product * GFP sequence * Regulation of pBAD promoter * Design Your Primer PCR of GFP (staining with Ethidium Bromide) [Student Guide] [Materials Guide] [Materials List] Extra material: * Sequencing the PCR Product * GFP sequence * Regulation of pBAD promoter * Design Your Primer ELISA assay How can you detect a viral disease, such as AIDS? Students perform a diagnostic test, the ELISA assay, to examine the spread of a simulated viral epidemic in a class. The assay detects which individuals are infected, and students apply their knowledge of immunology to understand how the assay works at the molecular level. By analyzing the classroom data, students determine the original carriers of the virus and examine how transmitted diseases spread in a population. [50 minutes plus 90 minutes] [Student Guide] [Teacher Guide] [Materials Guide] [Materials MSDS List] Muscle Protein Electrophoresis Almost all of the cells in your body have the exact same DNA, so how can all of the cells in your body look different? A cell must decide which DNA to use to make the proteins it needs to be that cell. For example, all muscle cells (skeletal, smooth, and cardiac) have both actin and myosin that help them contract, but the mechanism of contraction is different in different cells: cardiac and skeletal muscle use tropomyosin and smooth muscle doesn't. Since smooth muscle doesn't need tropomyosin to be able to contract, it doesn't make the tropomyosin protein. This activity allows students to understand that different genes are expressed in different tissues and therefore different proteins are present. [90 minutes plus 50 minutes] [Student Guide] [Teacher Guide] [Materials Guide] [Materials MSDS List] Protein Evolution Mutations in an organism's DNA can change its characteristics, and these characteristics can help the organism to survive and reproduce. Sometimes, organisms can change so much over many generations that their offspring become a new species. Some of their DNA and proteins will be very different, and some will be the same. Students can analyze muscle tissue from different species to correlate relatedness, by evaluating protein profiles and looking for proteins that are the same in all the species and proteins that are different. [90 minutes plus 50 minutes] [Student Guide] [Teacher Guide] [Materials Guide] [Materials MSDS List] Microarray Students will monitor the gene expression of numerous genes using a technique called microarray analysis. The class can analyze the difference in gene expression in breast caner tissue and compare that to non-cancer tissue. Students will learn about how cells control their expression of genes, what kinds of regulations are necessary and what genes and pathways are affected in cancer cells. Alternatively the class can analyze the difference in gene expression in the leaves of a plant that has been heat stressed versus not stressed. [can be done in 50 minutes -- 90 minutes to include discussion] [Student Guide] [Teacher Guide] [Materials Guide] [Materials MSDS List] [ Roots of Cancer.pdf, Hallmarks of Cancer.pdf ] Bacterial Evolution [Materials Guide] [Materials MSDS List] Water Sterility Students will test a varity of water samples for the presence of microorganisms. This lesson will teach how to use sterile technique, how to plate samples on to petri dishes and extrapolate the results to calculate the amount of bacteria in larger volumes of water. The lesson can include the sterilization and confirmation of sterilization of samples tested. [Student Guide] [Materials Guide] [Materials MSDS List] *************************************************************************************************************************************************************************************** For advanced BIOTECH activities (used primarily in Biotechnology courses) Click on activity name to download the activity sheet: Penicillium Antibiotic Effect How were antibiotics discovered? How is the effect of an antibiotic different for different species of bacteria? This activity touches on the history of early antibiotics research as well as serves as an example of how observation leads to discovery. We have optimized a set of experiments to fit the high school classroom working with an antibiotic producing fungus and species of bacteria to emulate early observations of antibiotic effect on bacteria. Students normalize cultures of Penicillium fungi (green bread mold) as well as bacterial species Staphylococcus epidermidis, Microcuccus luteus, and Enterobacter aerogenes using spectrophotometry before co-culturing the fungus with the bacteria to witness the antibiotic effect. Students then measure the results of co-cultivation by quantifying the optical density of the bacteria at the end of one week of experimentation. [5 days] [Student Guide] [Teacher Guide] [Materials Guide] [Penicillium Antibiotic Effect Page] [Materials MSDS List] SDS-Polyacrylamide Gel Electrophoresis w/o Bradford Assay SDS-PAGE is used to separate proteins between molecular mass of 5 and 250 KDa, and allows the student to visualize major proteins within tissues. This activity can be used to visualize that different genes are expressed in different tissues and therefore different proteins are present, using different tissues from a single animal (for example cow--skeletal, smooth, cardiac, kidney, liver tongue, cheek, and brain). Alternatively, students can analyze muscle tissue from different species to correlate relatedness, by evaluating protein profiles and looking for proteins that are the same in all the species and proteins that are different (for example different fish). [50 minutes for protein extraction, 90 minutes run gel plus 50 minutes for analysis] [Student Guide] [Materials Guide] [Materials MSDS List] SDS-Polyacrylamide Gel Electrophoresis w/ Bradford Assay In order to make any quantitative analysis of the results of SDS-PAGE, students need to load equivalent total protein for each of the protein samples on the gel. Bradford assay is used to quantify the total protein in each of the tissues prior to SDS-PAGE. The rest of the activity is the same as the description above. [50 minutes for protein extraction, 50 min for Bradford assay, 50 minutes for calculations and dilutions, 90 minutes run gel plus 50 minutes for analysis] [Student Guide] [Materials Guide] [Materials MSDS List] Western Blotting Take your students to the next level of protein analysis with Western Blot, and test for a specific protein in tissue. In this investigation students extract proteins from cow--skeletal, smooth, cardiac, kidney, liver tongue, cheek, and brain, determine the protein concentration of their extracts, separate proteins based on size on SDS-PAGE, transfer the proteins to nitrocellulose and probe with an antibody against alpha-actin. [Student Guide] [Materials Guide] [Materials MSDS List] Enzymatic Activity of Cellobiase (aka BioFuels from BioRad): One ml cuvette version Cellobiase is and enzyme involved in the last step of the process of breaking down cellulose, a molecule made up of bundled long chains of glucose that are found in plant cell walls, to glucose. This is a natural process that is used by many fungi as well as bacteria to produce glucose as a food source. Students will use fungi (white mushroom) to extract cellobiase and measure enzyme activity. Students can also alter enzyme activity but changing temperature, changing pH, and changing salt concentration. This lab activity lends itself for scientific method of making hypotheses and testing them. If your school has spectrophometers with cuvette holders then you can use this version of the lab. If you have the old Spec 20s and use a glass culture tube, then you will need the four ml version below. [Student Guide] [Materials Guide] [Materials MSDS List] PCR for 35S Promoter in Corn Students can grow either corn or soy bean which have been genetically modified along side a non modified control and use PCR to detect a piece of the GM DNA. Most GM plants use the strong constitutive 35S promoter, which will be used for PCR amplification to determine whether the plant has been modified. A control PCR will be amplified to verify that extracted DNA can be used for PCR, using Tubulin primers which is present in all plants. This activity allows students to understand the use of PCR as a detection tool, as well as the specificity of primers. [Student Guide] [Materials Guide] [Materials MSDS List] Extra reading for GMO lab activities: * [The New Breed] * [A Hard Look at GM Crops] Determine If Your Food is Genetically Modified Students can test various foods to determine if it has been genetically modified using the same PCR analysis in the above lab activity. Food sources with readily amplifiable GM DNA include most processed corn products, such as corn tortilla, tamales, and masa, and papaya. [Student Guide] [Materials Guide] [Materials MSDS List] DNA Barcoding of Insects DNA barcoding involves the use of a single gene to identify a given species through the comparison of nucleotide sequences in the DNA to that of the same gene in other species. Animal DNA barcoding involves sequencing a short fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Students can collect insects, extract DNA, amplify the COI gene, verify the presence of a PCR product and send the PCR product to be sequenced. Students can then compare the sequence to those in Genbank and determine specicies identity, as well as compare the class's insect sequences to each other and build a phylogenetic tree (see next lesson DNA Barcoding Sequence Analysis using DNA Subway). [Student Guide] [Materials Guide] [Materials MSDS List] Extra Reading DNA Barcoding activities * [Scientific American] * [Stoeckle] * [Herbert] Insect DNA Barcoding Sequence Analysis using DNA Subway Students will compare DNA sequences from insects to those in Genbank and determine species identity, as well as compare the sequences to each other and build a phylogenetic tree [Student Guide] [Materials Guide] Plant DNA Barcoding DNA barcoding involves the use of a single gene to identify a given species through the comparison of nucleotide sequences in the DNA to that of the same gene in other species. Plant DNA barcoding involves sequencing a short fragment of a chloroplast gene, Rubisco Large Subunit. Students can collect plant samples, extract DNA, amplify the RubL gene, verify the presence of a PCR product and send the PCR product to be sequenced. Students can then compare the sequence to those in Genbank and determine species identity, as well as compare the class's plant sequences to each other and build a phylogenetic tree (see next lesson Plant DNA Barcoding Sequence Analysis using DNA Subway). [Student Guide] [Materials Guide] Plant DNA Barcoding Sequence Analysis using DNA Subway Students will compare DNA sequences from plants to those in Genbank and determine species identity, as well as compare the sequences to each other and build a phylogenetic tree. [Student Guide] [Materials Guide] Comparing PCR of a Single Loci to Restriction Digest of the Entire Genome [Student Guide] [Materials Guide] [Materials MSDS List] Design Your Primer [Student Guide] C. elegans Mutant Genetic [Student Guide] [Materials Guide] [Materials MSDS List] Extra material for C. elegans Mutant Genetics: * [Nemotode Growth Media and Passaging of C. elegans] * [C. elegans Life Cycle] Identification of Bacterial Species [Student Guide] [Materials Guide] [Materials MSDS List] Extra Material: * Antibiotic Sensitivity Testing * Gram Staining Extra Reading * 16S rDNA Sequence Analysis Middle School Activities Kiwi DNA Extraction How do you purify DNA from cells? Students extract DNA from kiwifruit to learn about the chemical and physical properties of DNA. This activity provides a first-hand understanding of how DNA can be isolated for further analysis, such as DNA fingerprinting. Students also reinforce their understanding of cell structure and biological macromolecules. We use a kiwifruit protocol because it uses commonplace materials and requires little equipment. [45 minutes] [Student Guide] [Teacher Guide] [Materials Guide] [Materials MSDS List] DNA Fingerprinting How is DNA evidence prepared and analyzed in a crime case? Students perform agarose gel electrophoresis to analyze DNA (dye simulation) samples from a mock crime scene. Based on DNA fingerprinting profiles with dyes simulated to represent the DNA a comparison is made to the crime scene, students determine which suspect likely committed the crime. This activity helps students understand how DNA variation in individuals can be analyzed in practical applications such as genetic testing and forensics. [50 minutes to introduce electrophoresis and practice pipetting, 50 minutes to run gels, partial next day to analyze results] Some Examples: * Cat Food Caper - [Student Guide] [Teacher Guide] [Materials Guide] [Materials MSDS List] * Bubble Gum Mystery - [Student Guide] [Teacher Guide] [Materials Guide] [Materials MSDS List] * Romance Mystery - [Student Guide] [Teacher Guide] [Materials Guide] [Hair1][Hair2][Hair Analysis][Fingerprints][Types][Card] [Materials MSDS List] * Todd Family Paternity - [Student Guide] [Materials Guide] [Teacher Guide] [Materials MSDS List] Genetic Testing for the PTC Gene Jillian a student at Cactus High School in Peoria. Her middle school class learned about PTC tasting when her class learned about traits. As it turned out, she was not a taster. In high school, Jillian decided to get some PTC paper and have her family do the taste test, and draw a family tree based on the tasting data. Surprisingly, everyone in her family is a taster, her mother, her father, both her brothers, even her grandparents and aunt and uncle. Jillian was quite perplexed. Is it genetically possible that she is not a PTC taster? [One 50 min class period for introduction, 50 min gel electrophoresis, and partial class period follow-up and final analysis] [Student Guide][Teacher Guide] [Materials Guide] [Materials MSDS List] Sickle Cell Anemia A patient and his wife come in to see you with a concern. The patient has a history of sickle cell disease in his family, but neither of his parents have exhibited any symptoms. The wife is an immigrant from rural tropical Africa and has no idea if her family has any history of sickle cell disease. However the area she is from has a high incidence of sickle cell anemia in the population. The couple has 2 children, ages 4, 8 and would like to have another. Their kids don’t know about the history of the disease. The couple has come to you for advice on whether or not to have another child, and what to tell their children about the family medical history. [One 50 min class period for introduction, 50 min gel electrophoresis, and partial class period follow-up and final analysis] [Student Guide] [Materials Guide] [Teacher Guide] [Materials MSDS List] PTC--To Taste or Not to Taste Why do you think some students can taste the PTC and others can’t? Are you a taster or not? Students will test the DNA of Jillian and her family for the PTC gene and determine if their genetics correlates with the tasting data. The most common PTC gene mutation (resulting in the inability to taste PTC) in the US population is due to a deletion of part of the gene, which is easily tested for and visualized by DNA electrophoresis. Students will use this information to help them draw a family tree for Jillian. [Student Guide] [Materials Guide] [Materials MSDS List] Cootie Genetics In this activity students will simulate the work of Gregor Mendel to investigate how traits are inherited. Students mate "Cootie" organisms with different true breeding traits and explore trait behaviors (dominant, recessive) and trait probabilities- while having fun! This lesson should be introduced before genetic terminology, DNA and/or Punnett Squares. [Three to four 50 minute class periods] [Student Guide] [Materials Guide] [Click here to access Cooties web site] Disease Detection Students will simulate the outbreak of a viral disease in the classroom starting with one individual that is infected. They will analyze the classroom data, to determine the original carrier of the virus and examine how transmitted diseases spread in a population. [50 minutes] [Student Guide] [Teacher Guide] [Materials Guide] DNA Origami Learn more about DNA structure with this classic paper folding activity. This activity was designed by DNA Interactive (http://www.dnai.org), and has been slightly modified. Students will see that the backbone of DNA comprises of sugars and phosphates whereas the bases are on the inside of the structure, and they will see the antiparallel nature of DNA. They will learn that Adenine is always paired with Thymine, and Guanine always with Cytosine, and that AT base pairing uses two hydrogen bonds, where as GC uses three hydrogen bonds. [Origami Color, B&W, Instructions]