Team:UI Indonesia/InterLab
InterLab Studies
Introduction
In the field of engineering, repeatable and reproducible measurements are important to obtain valid and reliable results. However, these have been proven difficult to achieve as there are differences in environmental condition of laboratories, individuals conducting the measurements, instruments being used, and other sources of variability. Eventually, this could lead to hindrance of advancements in engineering, including synthetic biology.
For past several years, iGEM Measurement Committee has been working on this issue by encouraging registered iGEM 2018 teams to participate in annual InterLab study. Since its introduction in 2014, the study has been conducted four times, making this year’s study to be the fifth one. The goal of this study is to minimize possible sources of variability in laboratory measurements and thus allowing synthetic biology to attain its full potential as a tool for improving quality of life.
InterLab study is mainly focused on fluorescence measurements as one of widely utilized protocols in synthetic biology studies. Data obtained from such measurements are often reported in different units, or processed in different methods, thereby hampering fluorescence data comparison. Hence, iGEM Measurement Committee introduces a standardized protocol for green fluorescence protein (GFP) expression level measurement. Previous studies showed that variability of the measurements can be significantly reduced by calibrating measured absolute fluorescence units of expressed GFP against known concentration of florescent molecule. However, when the procedure is carried out against a population of cells, the cell number in given sample makes great variability among measurements. This is due to the values of total cell number used to calculate mean expressed GFP per cell are obtained from optical density (OD), which is a subject to large variability.
Therefore, in this year’s InterLab study, participating 2018 iGEM teams are encouraged to help iGEM Measurement Committee in investigating whether more direct method in expressing total cell number for fluorescence calculation, such as absolute cell count or colony-forming units (CFUs), are better than OD to reduce variability in bulk measurement. We proudly announce our participation in 5th InterLab study for the first time ever, in the hope that our results may contribute to the improvements in synthetic biology. Our members contributing in this InterLab study are shown in Figure 1.
Materials and Equipments
Some of the materials for this study are obtained from 2018 DNA Distribution Kit. Other materials and equipment are provided by Institute of Human Virology and Cancer Biology (IHVCB), UI.
Materials from 2018 DNA Distribution Kit
• LUDOX CL-X (45% colloidal silica suspension) stock
• Microspheres (silica beads suspension) stock ~ 4.7 x 108 microspheres
• Sodium fluorescein stock
• Devices (parts in pSB1C3 plasmid backbone, all dried in Distribution Kit Plate): negative control, positive control, test device 1, test device 2, test device 3, test device 4, test device 5, and test device 6.
Materials and equipment provided by IHVCB, UI:
• ddH2O
• 1x phosphate buffered saline (PBS), pH 7.4-7.6
• 96-well plate (clear plate with flat bottom)
• 96-well plate reader (GloMax®– Multi Detection System, Figure 2). Specifications: can measure both absorbance and fluorescence, no settings for pathlength correction and temperature adjustment, has four installed filters and two customizable filter holders in six-position filter wheel, reads the samples from top of the plate
• Competent cells of Escherichia coli strain DH5α
• Luria-Bertani (LB) media, liquid and agar
• Chloramphenicol (dissolved in absolute ethanol at concentration of 25 mg/mL, when added into LB media it should be at ratio 1:1000)
• 50 ml Falcon tubes
• Incubator (set at 37oC)
• 1.5 ml microtubes
• Bucket with ice
• Micropipettes and tips
Methods
Safety cautions
During laboratory works, we took extra precautions to minimize potential harm. We wore personal protective equipment (PPE) such as laboratory safety gowns with head cover, masks, and gloves (Figure 3, left). For procedures that require aseptic technique such as bacterial inoculation, we conducted the experiments in biosafety cabinet (BSC) to prevent contamination (Figure 3, right). We also managed every infectious waste according to our laboratory’s standard operating procedures, such as decontaminating all media used for bacterial growth with 10% sodium hypochlorite overnight before being thrown into appropriate container and autoclaving them (121oC, 20 minutes) along with other infectious wastes. In addition, to ensure validity of our results, we used same instrument settings, 96-well plates, and volumes of sample added to the plates in entire protocol whenever possible.
Part 1: Calibration
Before we proceeded to Part II and III of the protocol, we had made three calibrations as follows:
• Calibration #1: this calibration is aimed to obtain a conversion factor to transform absorbance at 600 nm wavelength (Abs600600 as would be measured with spectrophotometer due to Abs600 data are volume dependent. First, we made four replicates of LUDOX CL-X and ddH2O in 96-well plate (each well contains 100 uL volume). We then measured Abs600 of the samples and recorded them. The Abs600 data were calculated for their respective mean and then subtracted each other to obtain corrected Abs600. OD600 measured with spectrophotometer (from reference) was divided with corrected Abs600 to get the conversion factor.
• Calibration #2: this calibration is aimed to obtain particle (i.e. microspheres with similar properties to cells) standard curve so it can be used to estimate the cells number in given samples from their Abs600. First, we made microspheres stock solution by adding 904 uL ddH2O to vortexed 96 uL microspheres with known concentration. We prepared 100 uL ddH2O in each well from second until twelfth column, first until fourth row of 96-well plate. We also created four replicates from microspheres stock solution in the first column (each well contains 200 uL solution). We then made a serial dilution for each replicate by transferring 100 uL from first into second column, mixing it thoroughly, and so on until we discarded 100 uL from eleventh column (i.e. twelfth column only contain pure ddH2O). As soon as each well had been remixed, Abs600 of the samples were measured and recorded in Excel. (Figure 4)
• Calibration #3: this calibration is aimed to obtain fluorescence (i.e. fluorescein with similar properties to GFP) standard curve so it can be used to estimate fluorescence of cell-based measurements (in Part II) from equivalent fluorescein concentration. First, we made 1x fluorescein stock solution (10 uM) by diluting spun-down fluorescein in 1x PBS with appropriate volume. We then created four replicates of 1x fluorescein stock solution and serial dilution in 96-well plate similar with Calibration #2 protocol. Fluorescence for each sample were measured with “BLUE” settings in our plate reader: 490 nm excitation, and 510-570 nm emission. The data obtained were recorded in Excel. (Figure 5)
Part 2: Cell Measurements
This part of protocol is aimed to measure Abs600 and fluorescence of transformed Escherichia coli strain DH5α with devices provided from 2018 DNA Distribution Kit Plate. All devices are in plasmid pSB1C3, which carries a gene for chloramphenicol resistance. iGEM Registry of Standard Biological Parts (link) provides detailed information about devices being transformed in this study (Figure 6).
• Positive control contains promoter J23151 with GFP construct (part number: BBa_I20270).
• Negative control only contains TetR repressible promoter without GFP construct (part number: BBa_R0040).
• Test device 1 contains promoter J23101 with GFP construct (part number: BBa_J364000).
• Test device 2 contains promoter J23106 with GFP construct (part number: BBa_J364001).
• Test device 3 contains promoter J23117 with GFP construct (part number: BBa_J364002).
• Test device 4 contains promoter J23100 with GFP construct (part number: BBa_J364007).
• Test device 5 contains promoter J23104 with GFP construct (part number: BBa_J364008).
• Test device 6 contains promoter J23116 with GFP construct (part number: BBa_J364009).
The workflow for this part is shown in Figure 7. First, we transformed each device into Escherichia coli strain DH5α according to our laboratory’s protocol (full version please refer to this file) and subsequently plated into different LB agar with chloramphenicol (Cam). After overnight incubation at 37oC, two colonies from each transformation plates were inoculated in 10 mL LB liquid medium + Cam and let to be grown overnight at 37oC and 220 rotations per minute (rpm). On the next day, we diluted 0.5 mL of each culture in 4.5 mL LB + Cam (ratio 1:10) and measured for their respective Abs600, along with Abs600 of LB + Cam (we created two replicates to be measured for each sample). Using obtained Abs600 data, the cultures were further diluted to target Abs600 0.02 in final volume of 12 mL LB + Cam in different 50 mL Falcon tubes covered with foil. From each Falcon tube, 500 uL diluted cultures were sampled and measured for Abs600 and fluorescence, while the rest of diluted cultures were incubated at 37oC, 220 rpm until next six hours to be sampled and measured again. Samples for Abs600 and fluorescence measurements were put in 96-well plates (each well contains 100 uL volume) according to layout shown in Figure 8, under same conditions as previous calibration in Part I of the protocol.
Part 3: Colony Forming Units (CFUs)
This last part of protocol is aimed to convert OD600 (obtained after transformation of Abs600 from Part II with conversion factor from Part I) into CFUs. The significance of this part is that CFU counts are directly correlated with cell concentration in given volume of culture (i.e. viable cell counts per mL). In this part, we used two colonies from each positive and negative control (four in total). This part consists of three steps as follows:
• Step #1: Preparation of starting sample. First, we sampled 25 uL of each positive and negative overnight cultures and diluted them with 175 uL fresh LB + Cam (eight-fold dilution). We then measured their respective Abs600 along with blank LB + Cam according to layout shown in Figure 9, red inset. Using this data, their OD600 were calculated by multiplying each of them with conversion factor (obtained from Part I). The overnight cultures were further diluted to target OD600 0.1 using previous calculated OD600 data in final volume of 1 mL LB + Cam (these are “starting samples”). We then created triplicates of each starting sample (twelve in total) and checked for their OD600 if they were 0.1, using measurement with same 96-well plate (Figure 9, green inset).
• Step #2: Serial dilution of starting sample. Originally, we wanted to conduct exact workflow as shown in Figure 10. However, we realized the maximum capacity of our microtubes was 1.5 mL so we could not aliquot 1.9 mL LB + Cam in the tubes. Therefore, to compensate that issue, we first prepared 36 microtubes with 950 uL LB + Cam each, 24 microtubes with 900 uL LB + Cam each, and 36 LB agar + Cam. For first three dilutions, we halved the volume of LB + Cam in “Dilution 1” until “Dilution 3” tubes and the added volume of starting sample (50 uL into next 950 uL LB + Cam) so the net dilution factor would be the same (20-fold). Finally, we took 100 uL from each last three dilutions to be spread on different LB agar plate + Cam. The plates were then incubated overnight at 37oC and the colonies formed on each plate were counted after 18-20 hours of growth.
• Step #3: Calculation of CFU/mL for OD600 0.1. This calculation assumes that one bacterial cell gives rise to one colony. For OD600 0.1, CFU/mL culture can be calculated by multiplying number of colonies (we used plates with less than 300 colonies in Step #2) with their respective final dilution factors.
RESULTS AND DISCUSSIONS
For complete raw data of calibrations (in Part I) and Abs600 and fluorescence measurement (in Part II), please refer to our Excel file attached here.
RESULTS AND DISCUSSIONS