Difference between revisions of "Team:UI Indonesia/InterLab"

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 (121^oC, 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 37^oC, two colonies from each transformation plates were inoculated in 10 mL LB liquid medium + Cam and let to be grown overnight at 37^oC 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 37^oC, 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.