Name of Tool/Strategy: Producing Green Fluorescent Protein in Insect Cell Culture Using Baculovirus, A New Laboratory Experience for UC Davis Biochemical Engineering Students
Course Information:
Course Name: ECH 161L, Bioprocess Engineering Laboratory
Enrollment: 21
Brief Description: Laboratory experiments in the operation and analysis of bioreactors, the determination of oxygen mass transfer coefficients in bioreactors, and the use of ion exchange chromatography for protein separation.
Description of Tool/Strategy Implementation:
Insect-derived baculoviruses have emerged as safe and versatile expression vectors and are widely used as tools for recombinant protein product in insect cells. Expression Systems, a company headquartered in Davis, CA that specializes in baculovirus/host cell systems, generously donated materials with which a new undergraduate laboratory experience could be created for senior biochemical engineering students in the Bioprocess Engineering Laboratory course (ECH 161L). The goal of the new experiment was to produce a recombinant green fluorescent protein (GFP) expressing baculovirus and use it to infect Sf9 insect cells, derived from the Spodoptera frugiperda cell line. The students were required to report the infection efficiency, as quantified by the amount of GFP expressed, as a function of MOI (multiplicity of infection).
Background:
The Bioprocess Engineering Laboratory has traditionally consisted of three laboratory experiences. In the first laboratory, students measure the volumetric mass transfer coefficient, kLa, using a dynamic gassing out method. The goal of the experiments it to collect data on dissolved concentration (%DO) in water over time at a number of operating conditions (such as variable agitation rates and aeration rates) so that a correlation can be made between kLa and the operating parameters that were varied. Students receive their first experience with a laboratory scale bioreactor and associated control system interface during this lab as well as experience with a dissolved oxygen probe. In the next laboratory, students separate a mixture of three proteins (Cytochrome C, Myoglobin, and Bovine Serum Albumin) using anion exchange chromatography. The product samples are then characterized using SDS-PAGE. The goal of the lab is to determine the effectiveness of the separation and to quantify the recovery of the proteins. Students receive experience using an AKTA Explorer System as well as with SDS-PAGE during this laboratory experience. Pipetting skills are also taught in this lab. In the last of the traditional laboratory experiences offered in this course, students carry out a fermentation process using a genetically-modified strain of E.coli in order to produce green fluorescent protein (GFP). The goal of the experiment is for the students to determine the optimal glucose feeding strategy to obtain the highest GFP production, including the timing of the addition of the inducer (IPTG for this strain). Students learn a number of techniques in this lab including operating a bioreactor during a fermentation, using a spectrophotometer to measure the optical density (OD) of cells, using a YSI in order to measure glucose concentration, using a fluorometer to measure GFP expression, and making a cell dry weight vs. OD standard curve.
Summary of the Experiment:
As stated above, the goal of the new experiment was to produce a recombinant green fluorescent protein (GFP) expressing baculovirus and use it to infect Sf9 insect cells, derived from the Spodoptera frugiperda cell line. The students were required to report the infection efficiency, as quantified by the amount of GFP expressed, as a function of MOI (multiplicity of infection). This experiment was run in addition to the three traditional experiments previously described, and in parallel to the E.col fermentation experiment so that students were working on optimizing GFP production using two different expression systems. In the course of carrying out this new experiment, students gained experience with various techniques, potentially important for future employment in positions related to the biotech industry, that were not taught in previous iterations of the course. These techniques included working in a biosafety cabinet, measuring cell concentration using a hemocytometer, transfecting cells with recombinant viral DNA to produce viral progeny, and infecting cells with live virus.
The experiment was carried out over the course of three laboratory periods. On the first day, the students carried out a transfection of Sf9 insect cells with viral DNA. The viral DNA was designed to only produce viable virus if the gene for GFP was included. In order to carry out the transfection, students followed a protocol provided by Expression Systems. Work was primarily done in the biosafety cabinet and a hemocytometer was employed so that the students knew how much of the insect cell stock to use in order to plate approximately 0.9*105 cells. The transfected cells were incubated for five days so that virus would be produced and viral stocks could be harvested. While students waited between steps in the protocol, they prepared the solutions needed for the E.coli fermentation experiment. On the second laboratory day, the students harvested their recombinant baculovirus stocks. Half of the sample was used to infect new Sf9 cells in order to produce GFP, and the other half of the sample was sent to Expression Systems in order to determine the viral titer. This was done mid-day while the students were primarily focused on carrying out their E.coli fermentation. The students again worked with their cells and viral stocks in the biosafety cabinet, and the hemocytometer was again employed in order plate 0.9*105 new Sf9 cells. After these new cells were incubated with the student’s viral stocks for three days, the infected cells were visualized under the microscope and the amount of GFP present was quantified. Pictures of both uninfected and infected Sf9 cells are shown above. Infected cells swell and therefore should appear slightly larger than the uninfected cells. A fluorometer was employed to measure the amount of GFP in each of the students’ cultures. These results were used along with the viral titer results obtained from Expressions Systems in order to relate infection efficiency (expressed as GFP concentration) as a function of MOI.
The laboratory handout provided to the students can be found below. It is important to note that working with both baculovirus and Sf9 cells is very safe (BSL 1) as baculovirus does not infect human cells. No additional training beyond standard lab safety training was required. Students followed standard la safety procedures and also ensured that any cells/virus waste was either mixed with bleach or autoclaved.
Assessment and Analysis:
Informally, students have expressed excitement over the opportunity to work with this modern expression system and to gain experience working in the biosafety cabinet. Formally, the students were assigned to present an oral presentation detailing the background theory associated with this experiment, their experimental methods, and their results/analysis. One oral presentation was delivered per laboratory group which consisted of 3-4 students. Students were assessed based on the technical content in their report (background, theory, motivation, experimental methods, results, and discussion; accounted for 75% of the report grade) as well as on their slide composition and oral presentation skills (accounted for 25% of the report grade). Student performance on these oral presentations varied significantly. While it was required that each student participate in the transfection step of the experiment, some members of the “super-groups” (combination of two groups that worked on the E.coli fermentation lab together) took on more responsibility than others when it came to infecting the new Sf9 cells or measuring GFP expression. Because of this, it was obvious in the oral presentations that some students did not have a good concept of what was fundamentally occurring in the baculovirus/Sf9 system during the duration of the experiment. Further, only one of the three supergroups were able to obtain meaningful results. These can be seen on the figure below, where more GFP was observed in Sample 4, which has an MOI of approximately 0.1. Based on theory, it was hypothesized that the most product would be generated at this MOI. It is also clear that GFP is present in Samples 5 and 6, which had sub-optimal MOIs. One of the other two supergroups was also able to see some indications of GFP expression, but the amount of GFP in these samples as compared to the control was not appreciable. The third supergroup did not have any detectable virus in their viral stocks, as measured by Expression Systems. Further, as would be expected, negligible GFP expression was found. This was the first group to rotate through this lab, and the growth patterns of the Sf9 parent cultures did not appear to be healthy (most likely due to improper freezing after the cells were received). New Sf9 cells stocks were obtained from Expression Systems for the last two supergroups, and these groups were able to make virus as described above.
Acknowledgements:
We are very grateful to David Hedin and Expression Systems for generous donating the materials needed for this laboratory. We also thank Dr. Brooks Hayes for his time spent teaching us how to carry out the transfection as well as providing a guest lecture to our students.
Experiment (Appendix):
Protein Expression in Insect Cell Culture Using Baculovirus
Introduction
In this lab, you will be producing a recombinant baculovirus and using it to infect Sf9 insect cells, derived from the Spodoptera frugiperda cell line, in order to produce green fluorescent protein (GFP) product. Three days will be required to transfect Sf9 cells with the recombinant baculovirus, harvest the virus and infect new Sf9 cells, and measure the efficiency of the infection. This lab will be more guided than your other lab experiences in this course. Your goal will be to observe the infection efficiency (as quantified by the amount of GFP expressed) as a function of MOI (multiplicity of infection). You will be quantifying cell concentration using a hemocytometer, and GFP concentration using a fluorometer. Expression Systems, the generous sponsor of this experiment, will be quantifying the titer of your viral stocks. You will also have the opportunity to visualize infected and uninfected Sf9 cells under the microscope, and to take pictures in order to visualize the swelling that occurs in infected cells.
Laboratory Preparation
Review the literature pertaining to use of baculovirus in insect cell expression, including the resources cited at the end of this document. The Expression Systems website also has a helpful support page (http://expressionsystems.com/support/?section=administrative-support, click on Media or Cells). You will be carrying out many of these steps in a biosafety cabinet, and therefore should familiarize (or re-familiarize) yourself with sterile technique. This lab will be carried out at the same time you are carrying out the E.coli fermentation experiment, and therefore your group should carefully plan when all tasks for both experiments will be carried out and who is responsible for what task. At least two group members should be in the lab at all times for each task. This means that if you are planning to carry out a task for this lab and the E.coli fermentation simultaneously, there should be four members of your group in the lab. For the experimental plan, you don’t need to include every detail listed in the following experimental setup, but you should summarize the key steps and note where you plan to deviate from the procedure, if at all.
Experimental Methods
A) Day 1: Transfection (Start at 9am on assigned Thursday)
Note: Sf9 cells will be passed to fresh insect cell culture medium by the TA the evening before your experiment begins so that your cells are (hopefully) in early log phase. Cells will be kept in an incubator shaker @ 140 rpm and 27 °C. Gentamycin will be added to the media at a concentration of 10 μg/ml.
Using a 6-well plate, plate approximately 0.9*106 Sf9 cells (should be in log phase) in each well for each co-transfection. Use a microscope and hemocytometer in order to quantify the number of cells. Carry out one co-transfection per group member as well as one control reaction. Incubate the plate undisturbed at room temperature for 30 minutes to allow the cells to adhere.
While the plate is incubating, you may begin to prepare the co-transfection mixtures. For each co-transfection, pipiette 100 μL of transfection medium into two sterile polystyrene tubes. In one of the tubes, add 5 μL (0.5 μg) of BestBac DNA and 2 μg of plasmid with the GFP gene. In the other tube, add 6 μL of Expres2TR transfection reagent. Incubate both tubes at room temperature for 5 minutes before combining the two solutions in one tube. Mix gently by slowly pipetting up and down two times, using a 1000 μL pipette tip. Incubate this mixture at room temperature for 20 minutes.
Add 800 μL of transfection medium to each transfection mixture to increase the volume to 1000 μL. Aspirate the cell culture medium from each well and add the transfection solution to the appropriate well. To prevent drying of the monolayer, only aspirate and add transfection mixture to one well at a time.
Incubate the plate at 27 °C for 4-5 hours in a plastic bag or other sealed container with a damp paper towel or reservoir of water to maintain a humid environment.
After the incubation period, aspirate the transfection solution and immediately replace with fresh cell culture medium. Antibiotics (gentamycin, 10 μg/ml) may be added at this point if desired. Incubate the plate at 27 °C for 4-5 days in a plastic bag or other sealed container with a damp paper towel or reservoir of water to maintain a humid environment.
B) Day 2: Viral Harvest + Infect New Cells (on assigned Tuesday around 1pm)
After the 4-5 day incubation period, harvest the virus by transferring the supernatant to sterile centrifuge tubes. Use your viral stocks to infect new samples of Sf9 cells (you will need to start an overnight culture the day before you do this step). A sample of each viral stock will be sent to Expression Systems in order to quantify the viral titer. You may choose to experiment with different concentrations of cells, as the MOI will depend on both the titer of the virus and the concentration of the cells. Follow the plating procedure outlined in the Day 1 procedure to fix the cells, and add the viral stocks in lieu of the transfection medium. Prior to infecting the cells, view these uninfected cells under the microscope, take a picture, and note your observations regarding the size of the cells. Incubate the cells infected with the virus for approximately 72 hours at 27 °C.
C) Day 3: Visualize Infection and Quantify Infection using GFP Marker (1pm-2pm on assigned Friday)
Visualize the infected cells using the microscope and take a picture. The cells should appear slightly bigger than the uninfected cells that you viewed a few days ago. Quantify the amount of GFP that is being expressed using the fluorometer. The GFP concentration can be used to quantify the efficiency of the infection.
Suggested Components of the Report
In your report you should present an overview of your experimental method including photos/figures of key steps. Show the pictures you took of the samples under the microscope and comment on if you were able to visualize the infection (swelling of the cells). You also should define the concentration of cells used in each trial and the GFP expressed in each culture (a measure of baculovirus infection efficiency) as a function of MOI. Comment on the level of expression as well as possible extensions to this lab.
Safety
Standard safe laboratory procedures apply to this laboratory. Read all MSDS sheets for any chemicals you plan to use before beginning the experiment and make notes of any hazardous materials and/or steps in your lab notebook. Make sure that you use the proper personal protection (e.g. gloves, safety glasses) and disposal methods. Cells and virus must be bleached or autoclaved first before being disposed of down the drain. Closed toe shoes, lab safety glasses and long pants must be worn in the lab at all times and food/drinks are not allowed in the lab. Check with the TA before disposing of anything (either down the drain or in the garbage).
References
Airenne, Kari, et al. “Baculovirus: An Insect-derived Vector for Diverse Gene Transfer Applications.” Molecular Therapy 21(4):739-749, 2013.
“BestBac™ Linearized Baculovirus DNA Instructions for Use.” Expression Systems, Doc ID: 34524, Rev. No. 2, 2016.
O’Reilly, David R., Miller, Lois, and Luckow, Verne A. Baculovirus expression vectors: a laboratory manual. W.H. Freeman, New York, 1992, 347p.
Van Oers, Monique M., Pijlman, Gorben P., and Vlak, Just M. “Thirty years of baculovirus – insect cell protein expression: from dark horse to mainstream technology.” Journal of General Virology 96:6-23, 2015.