Team:Tuebingen/InterLab

iGem Tübingen 2017

Team Inspiration Results Human Practice Lab Attributions

the fourth interlab study

Introduction

How similar can fluorescence measurements be if the same protocol is used all over the world? This question will be answered in the fourth International InterLab Measurement Study for iGEM 2017. For researchers it is important to standardize protocols to produce reproducible data. Fluorescence values measured from GFP and other fluorochromes are usually difficult to compare as different devices and methods give different values in different units. This year's InterLab Study focusses on a comparable measurement of fluorescence by establishing a step by step protocol for plate readers used by all iGEM teams.

Theoretical background

Teams are provided with the same protocol to measure GFP fluorescence with a plate reader. Eight different devices were tested which are listed below:

All test devices are composite parts containing GFP with constitutive promoters, a negative control without GFP is also included. The vector pSB1C3 has a chloramphenicol resistance. Device 4, 5, and 6 additionally contain a Bicistronic Design Element Number 2 designed by Mutalik 2013 (Mutalik et al.2013) . This element should induce precise and reliable gene expression.
For normalization standard curves were made with the provided measurement kit from iGEM.

Practical workflow

Before the actual measurement, calibration was performed for OD600 and a fluorescence standard curve was determined using a clear bottom black 96-well plate in four replicates.

Table 1: Instrument settings for calibration
Instrument Settings	OD6000 reference point LUDOX-S40	fluorescein fluorescence standard curve
Positioning delay	0.5 s	0.2
Number of flashes per well	20	25
Orbital/pathlength correction	off	off
Optic	top	top
gain		700
Excitation		485
Emission		520
Orbital/pathlength correction	off	off

Figure 1: Fluorescein standard curve obtained by dilution series of fluorescein in 4 replicates.

Subsequently, we performed , the actual measurement of 8 different devices as shown in figure 2.
First, plasmids were transformed in DH5-alpha using the standard transformation protocol from iGEM with the deviation of using LB medium instead of SOC medium.
(Further information on the used protocol).
Two colonies were picked for each device and incubated in 5-10 mL LB medium + Chloramphenicol (25 µg/mL). The next day the solution was diluted to an OD of 0.02 and 500 µL of the samples were taken and hold on ice at t=0, 2, 4, 6 h. Absorbance (OD600) and fluorescence were then measured using the FLUOstar OPTIMA from BMG LABTECH.
(For detailed protocol click here.)

Results and discussion

The provided protocol by iGEM was easy to implement by providing a step by step guide to perform the experiments.
Although our data has a high variance between the devices and between the replicates after normalization, device 1 and 2 showed significant higher fluorescence than device 3. This is in line with the data from the device’s reference in the Registry where device 1 was shown to have the highest absorption followed by device 2 and then device 3.

Table 2: Variant RFP with corresponding absorption values
	Variant RFP	Absorption / AU oder mAU
Device 1	J23101	1791
Device 2	J23106	1185
Device 3	J23117	162

Device 4, 5 and 6 with the Bicistronic Design Element Number 2 showed no real difference in comparison to device 1, 2 and 3 where this element was not present. When the data from all teams is compared we will see if there is a bigger influence on gene expression due to the different promoters used.
At time point 2 h the fluorescence signal was the highest despite for the positive control. If the expression of RFP induces stress, one explanation might be that the bacteria induce expression of proteases or reduce the amount of the necessary transcription factors.

Figure 3: Results show in µM Fluorescein/OD600 for Devices 1, 2, 3 in comparison to devices 4, 5, 6. Samples were taken at t=0, 2, 4, 6 h. Values smaller than 0 were excluded in the graphic. Biological duplicates are represented from each device. BCD2: Bicistronic Design Element Number 2.

References

Mutalik, V. K., Guimaraes, J. C., Cambray, G., Lam, C., Christoffersen, M. J., Mai, Q. A., ... & Endy, D. (2013). Precise and reliable gene expression via standard transcription and translation initiation elements. Nature methods, 10(4), 354-360.