INTRODUCTION

This year, the iGEM Measurement Committee offers to all teams the possibility to do the Fifth International InterLaboratory Measurement Study in synthetic biology. This work concerns the reliability and repeatability of scientific measurements.

This project involves providing the same protocols and gathering all data from different teams to build a database with reference values.

During this edition, the main objective is to enhance measurements precision in synthetic biology by detecting and correcting sources of errors. Last year, the goal was to reduce variability in fluorescence measurements (GFP) with a normalization of optical density (OD). This year, we tried to reduce this variability between labs by normalizing to absolute colony-forming units (CFUs).
The iGEM Sorbonne Université team gave us non-competent DH5-α strain. See the collaboration here.

DEVICES

For this study, we had to transform six plasmids and two controls. We used a protocol of thermocompetence by CaCl2.

Negative control BBa_R0040: sequence for pTet inverting regulator, corresponding to TetR repressible promoter.

Positive control BBa_I20270: promoter and GFP sequence.

And six GFP expressing constitutive devices:

Device 1 BBa_J364000
Device 2 BBa_J364001
Device 3 BBa_J364002
Device 4 BBa_J364007
Device 5 BBa_J364008
Device 6 BBa_J364009.

FIRST APPROACH

The first approach consists in a conversion between cells absorbance to absorbance of a known concentration of beads.

Absorbance of a known concentration of beads

First, we did a calibration, using LUDOX CL-X, to obtain a conversion factor (Figure 1). This factor enables us to transform absorbance data from our plate reader into a basic OD measurement which can be found in a spectrophotometer.

	LUDOX CL-X	H₂O
Replicate 1	0.085	0.069
Replicate 2	0.085	0.066
Replicate 3	0.086	0.064
Replicate 4	0.090	0.064
Arith. Mean	0.087	0.066
Corrected Abs600	0.022	/
Reference OD600	0.063	/
OD600/Abs600	2.930	/

Figure 1: Results of the first calibration. The experiment was performed 4 times. The row colored in red shows our conversion factor

Then, we carried out a second calibration, using silica beads in a microsphere suspension, to convert absorbance measurements into a number of cells. This conversion is based on the plotting of a standard curve of particle concentration (Figure 2) that we have determined during this calibration.

Figure 2: Our particle standard curve based on silica beads measurements.

We did the same work creating a standard curve of fluorescence for fluorescein concentration. Then, we had to use this to transform our cell based readings into a fluorescein concentration.

Figure 3: Our fluorescein standard curve based on fluorescence measurements.

Absorbance of cells

After calibrations, we began cell culture expression measurement. For that, we did overnight cultures of two colonies for each device. 24 hours later, we measured absorbance and fluorescence of growing cultures with our plate reader. Then, we used our fluorescence standard curve to transform our cell measurements into fluorescein concentrations (Figure 4).

Hour 0

Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
104	165	496	237	-13	444	237	216
35	129	457	252	-50	381	137	150
39	111	504	197	-51	433	209	156
-24	118	426	156	-17	-92	156	170
47	149	612	279	-40	581	240	177
61	122	588	258	-34	564	166	132
61	138	604	281	-44	576	224	156
59	119	559	239	-27	524	154	112

Hour 6

Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
256	2874	8585	3577	175	13888	1562	2137
247	2800	8638	3645	193	14119	1481	2142
265	2874	8618	3729	180	14152	1532	2024
177	2803	8627	3587	72	13714	1526	2057
211	3158	9642	4127	193	12911	1275	1784
234	3272	9661	4206	209	13103	1352	1828
222	3224	9749	4032	203	13199	1354	1862
185	3149	9730	4155	177	12921	1327	1798

Figure 4: Results for fluorescence per particle at hour 0 and hour 6.

According to those results, we can deduce the strength of each promoter.
Device 1 (BBa_J364000): high strength
Device 2 (BBa_J364001): high strength
Device 3 (BBa_J364002): low strength
Device 4 (BBa_J364007): high strength
Device 5 (BBa_J364008): medium strength
Device 6 (BBa_J364009): medium strength

SECOND APPROACH

This second approach involves counting how many colonies grow on a plate. With this number, we have determined a cell concentration for each sample. Then, with CFU values for positive and negative control samples, we calculated a conversion factor from absorbance to CFU.

Example for a final dilution factor of 8.10⁵

First, we counted the colonies on each plate.

Sample	Number of colonies
1.1	34
1.2	73
1.3	93
2.1	137
2.2	71
2.3	64
3.1	44
3.2	40
3.3	65

Then, we multiplied the colony count by the final dilution factor. Doing so, we obtained the CFU/mL.

CONCLUSION

The Interlab experience enabled us to understand the variability of values within machines. Students from Chimie ParisTech performed the Interlab and therefore they discovered new biological methods with the Interlab. Our results were accepted and we hope they will help researchers worldwide to better deal with variability through reading this publication and the results from the other labs.

Team:Pasteur Paris/InterLab