The fourth international InterLab Study has two main objectives: to establish a protocol for measuring Green Fluorescent Protein (GFP) that can be used in all labs around the world with access to a plate reader, and test if devices with ribosomal binding sites (RBS) make gene expression more precise and reliable as intended.
For this study we transformed six different plasmids, along with a negative and positive control into E. coli K-12 DH5-alpha. The plasmids, from here on referred to as devices, contain a constitutive Anderson promoter with low (Bba_J23117), medium (Bba_J23106) or high (Bba_J23101) strengths. The test devices express GFP (BBa_E0040) as a reporter in the pSB1C3 backbone. The plasmids of test device 1, 2 and 3 have a different ribosomal binding site (BBa_B0034) then the plasmids of test device 4, 5 and 6 (Figure 1, see refBBa_J364100). A plasmid containing a promoter and GFP sequence (Bba_I20270) and a plasmid containing a TetR repressible promoter (Bba_R0040) in the pSB1C3 backbone, were used as positive and negative control. All plasmids were delivered to us with the iGEM distribution kit.
Figure 1: Schematic overview of test devices. The test devices contain a constitutive Anderson promoter with low, medium or high strengths and express GFP as a reporter in the pSB1C3 backbone. Test devices 1, 2 and 3 have a different ribosomal binding site than 4, 5 and 6. The plasmids where transformed into DH5-α.
Information about the devices:
- Positive Control (BBa_I20270)
- Negative Control (BBa_R0040)
- Test Device 1 (BBa_J364000)
- Test Device 2 (BBa_J364001)
- Test Device 3 (BBa_J364002)
- Test Device 4 (BBa_J364003)
- Test Device 5 (BBa_J364004)
- Test Device 6 (BBa_J364005)
We made overnight cultures of two colonies of each transformation plate. The following day, the OD600 values of the overnight cultures were measured, diluted to a target OD600 of 0.02 and grown. Afterwards, the absorbance (600 nm) and fluorescence (485/520, gain = 100) of the growing cultures were measured in a 96 wells plate in a plate reader (Synergy H1, BioTek) at 0, 2, 4 and 6 hours.
We measured Abs600 of LUDOX to use LUDOX-S40 as reference to obtain a ratiometric conversion factor. By multiplying this factor with the raw Abs600 measurement data, we could transform our absorbance data into a standard OD600 measurement, accounting for instrument differences.
To make the standard curve of fluorescence for fluorescein concentration (Figure 2), we made dilution series of fluorescein in a 96 well plate and measured fluorescence with the same settings as the cell measurements. With this standard curve we could correct our cell measurements to an equivalent fluorescein concentration, ultimately converting this into a concentration of GFP.
Figure 2: Fluorescein fluorescence calibration curve. The fluorescein concentration is plotted against the measured fluorescence intensity of fluorescein. With this curve we converted the fluorescence of our cell measurements to a GFP concentration.
Figure 3: Concentration GFP. (a) Concentration GFP/OD600 over time of test devices 1, 2 and 3, positive and negative control. (b) Concentration GFP/OD600 over time of test devices 4, 5 and 6, positive and negative control.
Comparing the devices with the same ribosomal binding site (1, 2, 3 and 4, 5, 6), the different promoter strengths are reflected in our results. We see that the promoter in 1 and 4 is indeed the strong promoter as the GFP expression seems higher than the GFP expression in test device 2 and 5, respectively. The medium strength of the promoter in test device 2 and 5 shows the same trend in comparison to test device 3 and 6. However, no GFP expression in test device 3 and 6 was observed. The ribosomal binding site of test device 4, 5 and 6 seem to reduce the expression of GFP relative to the RBS in test device 1, 2 and 3.