InterLab
Background
“All of the 2016 iGEM teams are invited and encouraged to participate in the Third International InterLaboratory Measurement Study in synthetic biology.” Our team took part in this study with the aimed to standardize the measurements of fluorescence in different labs. The main task was to quantify expression of GFP in common, comparable or absolute units. In our case, we measured fluorescence using plate reader.
Design
Fluorescence is widely used as a proxy for promoter activity by expressing fluorescent proteins such as green fluorescent protein (GFP). Despite this is an indirect measurement, it provides a useful insight into expression levels and has significant advantage that it could be a real-time monitor without disrupting cells.
Fluorescence/OD600 is routinely used to give an adjustment of the relative expression per cell.
Morever, we aim to contain instrument variability, at least to some degree, by measuring a standard scattering solution of a mono-dispersed silica suspension (LUDOX). The objective is to see if a simple, single fixed-point measurement can be used as a ratiometric adjustment to provide greater uniformity in fluorescence/OD600 measurements across sites.
Material and Methods
Plasmids used
• Plasmid DNA (100 pg/uL in 10uL of Buffer EB)
o Test Device 1: J23101+I13504
o Test Device 2: J23106+I13504
o Test Device 3: J23117+I13504
o Test Device 4: J23101.BCD2.E0040.B0015
o Test Device 5: J23106.BCD2.E0040.B0015
o Test Device 6: J23117.BCD2.E0040.B0015
Strain used
• Escherichia coli TOP10
Materials
• Fluorecein Standard: one tube with dried down FITC for creating a Fluorecein standard
• LUDOX: one tube with 30% colloidal silica suspended in 1mL of water
• 1xPBS (phosphate buffered saline)
• Fluorecein Standard: one tube with dried down FITC for creating a Fluorecein standard
• Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
• 50 ml Falcon tube (or equivalent) or 250 ml shake flask for cell growth
• 1.5 ml eppendorf tubes for sample storage
• Ice bucket with ice
• Pipettes
• 96 well plate (Black with flat bottom)
Machines
• Thermo VARIOSKAN FLASH
• MAPADA UV-3100PC SPECTROPHOTOMETER
• HONOUR INCURATOR SHAKER
Software
• Microsoft Excel
Methods
• Calibration
o OD600 Reference point
o Fluorescein standard curve
• Cell measurement
o Transformation
o Measurements
Description
Figure 1. Enigneering E.coli TOP10 with different devices cultured on plates. From left to right: positive control, negative control, E.coli TOP10 transformed with device 1 to 6.
Result
OD600 Reference Point
Table 1. OD600 Reference Point
Fluorescein Standard Curve
Table 2. Original data of Fluorescein standard curve
Figure 2. Fluorescein standard curve.
Cell measurement
Figure 3. Fluorescence Intensity of E.coli transformed with Device 1 to 6 and two control groups. Intensities was measured under OD481 using Plate Reader per 2 hours.
As shown in Fig.3, the combination of RBS and promoter in Device exhibited the strongest fluorescent intensity in 6h. Among these six devices, Device 6 almost had no fluorescent intensity.
Original data link:
Figure 4. Bacterial concentration of E.coli transformed with Device1-6, Negative Control & Positive Control. Intensities were measured under OD600 using Plate Reader per 2 hours.
As shown in Fig.4, except Device 1, the concentrations of E.coli transformed with another five devices were almost the same. It demonstrated that the expression of GFP in E.coli with Device 1 might have some passive effect on cell growth.
Original data link:
Figure 5. Fluorescence intensity in each cell of Device1-6, Negative Control and Positive Control.
From the result in Fig.5, E. coli transformed with Device1 exhibited the strongest fluorescent intensity in each cell, which means the effect of promoter and RBS in Device 1 is the strongest to induce the expression of GFP. In contrast, the combined effect of promoter and RBS in Device 6 had no ability to express GFP, though E.coli can still grow vigorously.
Original data link:
https://static.igem.org/mediawiki/2017/9/9b/SHSBNU_17_14a07.xls
