In science it is important that studies are reliable and repeatable. However, in synthetic biology studies fluorescence results are often reported in different units or as a 'relative expression'.
To combat this, the 4th edition of the interlab is aimed to compare the absolute values using a detailed protocol and data analysis for GFP. The interlab study of 2014 and 2015 resulted in a ratio metric comparison between the fluorescence measurements of strong, medium and weak promoters in different E. coli strains. They concluded that ratios mostly depend on the choice of instrument and only partly on the E. coli strain used. Compared to previous years, this year’s protocol was more strict in several aspects, one of which include that the DH5-α E. coli strain needed to be used. For more information visit the iGEM interlab website.
Test devices
This year we were provided with 6 test devices, a positive and a negative control. All of these had chloramphenicol resistance built in a pSB1C3 vector. Additionally, all devices except the negative control had a promoter site, a ribosome binding site, GFP and two terminators built in, while the negative control only has the Tet R promoter and does not express a fluorescent protein. The GFP part (E0040) was the same in all samples, but there were different variants of the promoters and the ribosome binding sites. The three promoters of the test devices are from the Anderson family and have different strengths. Bba_J23101 has a high expression rate, Bba_J23106 a medium and Bba_J23117 a low low expression rate. The Ribosome Binding Sites (RBS) are based on the Elowitz repressilator (B0034), the Bicistronic Design Element Number 2 (BCD2, J364100), and a relative weak1 RBS based on Ron Weiss thesis (B0032).
Device
Backbone
Promotor
RBS
Protein
Terminator 1
Terminator 2
Test 1
pSB1C3
J23101
B0034
E0040
B0010
B0012
Test 2
pSB1C3
J23106
B0034
E0040
B0010
B0012
Test 3
pSB1C3
J23177
B0034
E0040
B0010
B0012
Test 4
pSB1C3
J23101
J364100
E0040
B0010
B0012
Test 5
pSB1C3
J23106
J364100
E0040
B0010
B0012
Test 6
pSB1C3
J23117
J364100
E0040
B0010
B0012
Positive
pSB1C3
J364100
B0032
E0040
B0010
B0012
Negative
pSB1C3
R0040
Methods
The host for each device was E. coli strain DH5-α, as specified in the interlab requirements. DH5-α was probably chosen as this strain is the most used E. coli strain for routine cloning applications, which makes it ideal for an interlab study. However, in our lab, we use the E. coli strain BL21(DE3), so we had to order the DH5-α specifically for the interlab study. Because we had no experience with the strain, we had to optimize the transformation protocol before we could start with the interlab measurements.
Next to the transformation of the plasmids into the right E. coli strain, calibration protocols needed to be followed. To be certain that the cell density at the start of the measurements are the same, it was needed to calibrate our plate reader for OD600. To obtain this, LUDOX 40 (provided by the iGEM HQ) was used as a single point reference for a ratiometric conversion vector, which we could later be used to transform the absorbance data into a standard OD600 measurement. This allowed us to quickly determine the OD600 after a single plate reader experiment.
Next to calibrating the absorbance at 600 nm, a fluorescence standard curve needed to be obtained. This was done with a dilution series of Fluorescein (also provided by the iGEM HQ) and measuring it in the standard modes of the plate reader. The standard curve resulting from the calibration measurements makes it possible to convert the measurement data into a concentration of GFP.
Results
Below are a table of the OD600 calibration with LUDOX and a figure with the fluorescein standard curve. The OD600 measurement shows a clear difference between H20 and LUDOX giving a conversion factor of 0.236.
Table 1: OD600 calibration measurement with Ludox and H2O
LUDOX-HS40
H2O
Replicate 1
0.0452
0.0339
Replicate 2
0.0452
0.0338
Replicate 3
0.0403
0.0317
Replicate 4
0.0434
0.0346
Arith. Mean
0.0435
0.0335
Corrected Abs600
0.0100
Reference OD600
0.0425
OD600/Abs600
4.24
Figure 1: Fluorescein standard curve with serial dilution at excitation 485 nm and emission at 530 nm
Hereafter the fluorescence and absorbance of all our cells were measured with the same settings as the absorbance and the Fluorescein. This to ensure that all the data is comparable.
Figure 2: The fluorescence of the different samples at the different OD's
Discussion
What can be seen in figure 1 is that test device 1 and 4 rise very fast, but their OD does not increase as far as the rest. Device 2 and 5 rise slower but have a higher OD while devices 3 and 6 have a high OD but little to no fluorescence. This was to be expected since the stronger the promoter the lower the cell growth. However, the influence of the Ribosome binding site can also be seen. The B0034 binding site seems to have a higher expression which is especially visible in the difference between device 2 and 5.
From the results presented in Figure 2 we can see that there is a dependency of the fluorescence and OD600 on the promoters and the RBS. Test devices 1, 2 and 3 have the same RBS but different promoters. The promoter of device 1 results in a strong expression, while the promoter of device 3 has barely any expression and the promoter of device 2 lies somewhere in between with a moderate expression. When comparing the RBS, we see that device 5, which has the same promoter as device 2 but another RBS, has a much lower expression. The same RBS is used in devices 4 and 6 and also resulted in a lower expression, while not as clear as the difference between device 2 and 4.
Furthermore, when looking at the increase of the OD600, which is an indication of the multiplication of bacteria, there seems to be a pattern. In the cases of low protein expression, the bacteria seem to multiplicate more, and the bacteria which are busy with expressing proteins are not multiplying very fast. This can be explained by the fact that protein expression takes effort and energy of the bacteria, which is also needed for the multiplication, meaning that bacteria that are busy with protein expression, will be less busy with multiplication.
From these results we can conclude that J23101 is the strongest promoter, followed by J23106 and J23177 however the expression of J23106 is higher since the cells proliferate more with this promoter. The strongest RBS seems to be B0034 since all test devices from this RBS have a lower fluorescence than the previous group and the OD did not change much.
