THE INTERLAB STUDY

In synthetic biology, the use of reporter genes, such as the ones coding fluorescent proteins, is common to test genetic constructions or to establish proofs of concept for instance. However, the method used to analyse fluorescence data can change between laboratories. As standardization and repeatability are two fundamental principles in synthetic biology, the iGEM Measurement Committee proposed to develop universal tools that would enable to compare data and reduce the variability between laboratories in fluorescence measurements. To do so, they ask every year iGEM teams to test tools and hypotheses. Because many iGEM teams usually take part in this study, they can provide conclusions supported by a strong statistical analysis. Impressed by this initiative, the team iGEM Evry Paris-Saclay decided to take part in this study once again this year.

Fluorescent measurements are usually normalized according to the cell density to allow comparisons. The turbidity of cell suspensions or optical density measured at 600nm is the marker used to assess cell density. Nonetheless, it appears that this normalization leads to the introduction of more variability between laboratories in the data, coming from the optical density at 600nm measurement. There are more accurate methods that can directly estimate the absolute cell count in a sample. This year, the iGEM Measurement Committee thus wondered if by using these methods and by normalizing fluorescent measurements according to the absolute cell count instead of the optical density at 600nm, the variability between the laboratories would be decreased. To do so, two approaches will be set up : (i) the first one aims to estimate the absolute cell count by converting absorbance at 600nm of cell suspensions to absorbance at 600nm of a known silica beads that have similar physical properties as E. coli cells, creating then a universal standard ; (ii) the second one aims to directly count the number of colony-forming units in each sample.

MATERIALS

The plate reader used is the CLARIOstar from BMGLabtech (Figure 1) and the strain used is E. coli DH5α from New English Biolabs (NEB). The measurements were performed in a black 96-well polystyrene microplate with flat bottom from Corning. The silica beads, the colloidal silica suspension (LUDOX) and the fluorescein come from the 2018 iGEM distribution kit.

SAFETY

Labcoats, long pants, gloves and a dometic biological safety cabinet were used during these experiments.

CALIBRATION

The protocol summarised in Table 1 was used for the fluorescence intensity and the absorbance measurements by the CLARIOstar. All measurements were performed at room temperature (26.9°C).

Calibration 1: reference point

Absorbance measurement depend on the optical path. On a spectrophotometer, it is defined by the length of the cuvette, but on a plate reader, it varies depending on the volume in each well. In order to convert absorbance measurements from a plate reader to the ones from a spectrophotometer, the absorbance at 600nm from a 45% colloidal silica suspension (LUDOX) was measured by a plate reader (4 replicates were performed) (Table 2).

Calibration 2: particle standard curve

The aim here is to use different dilutions of a suspension of a known concentration of silica beads that share similar physical properties to E. coli cells to establish a particle standard curve.

After checking that the provided silica beads suspension hasn’t been frozen, 96 µL of it was added in 904 µL of milliQ water in an 1.5 mL Eppendorf tube. The tube was then vortexed for 10 seconds and 10 serial dilutions by a factor 2 were realised in a microplate in a final volume of 100 µL. The last well was filled with 100 µL of water. 4 replicates were performed.

The protocol is depicted in Figure 2 and the results presented in Figures 3 and 4.

Calibration 3: Fluorescence standard curve

Because plate readers measure fluorescence intensity in arbitrary units, it is necessary to establish a fluorescence standard curve to compare values from different plate readers.

To do so, a 10X fluorescein solution is prepared with PBS in 1 mL. Then a 1X solution, at 10 µM is prepared in 1 mL with PBS too. Ten serial dilutions by a factor 2 were realised in a microplate in a final volume of 100 µL. The last well was filled with 100 µL of PBS. 4 replicates were performed.

The protocol is depicted in Figure 5 and the results presented in Figures 6 and 7.

CELL MEASUREMENTS

Transformations

Competent E. coli DH5α ware transformed with test devices 1 to 6, as well as with a positive and a negative control (Table 3), following the iGEM protocol : http://parts.igem.org/Help:Protocols/Transformation.

Two clones for each constructs were then isolated and cultured in liquid media at 37°C under agitation (220 rpm) overnight (Figure 8).

Measurements
The cultures were diluted in LB (Luria Bertani) supplemented with chloramphenicol by a factor 10 in 5mL. The absorbance at 600nm was measured in a microplate. Then, they were diluted again in order to have cell suspensions at a absorbance at 600nm of 0.02 in 12mL in a 50mL Falcon tube :

500µL of each cultures were collected in 1.5mL Eppendorf tubes that were placed on ice. The rest of the cultures were incubated at 37°C and under agitation (220rpm) during 6h. Two microplates were then prepared according to the following layout (one for the t=0h samples and one for the t=6h samples). 100µL per well were deposited.

Colony forming units per 0.1 OD600nm E. coli cultures

E. coli DH5α + neg. control and E. coli DH5α + pos. control cultures are diluted by a factor 8 in 200µL of LB supplemented with chloramphenicol. The OD600nm is measured in a microplate. The measurements are performed in duplicate :

These samples are then diluted in order to obtain 3 cell suspensions per sample with an OD600nm of 0.1 in 1mL :

The OD600nm of the diluted samples was then measured to check that it equals 0.1. 200µL of each diluted sample were deposit in a microplate. These cell suspensions were then diluted by a factor 8*103, 8*104 and 8*105 according to the following scheme and then 100µL were plate on petri dishes containing 20mL of LB agar supplemented with chloramphenicol at a final concentration of 35µg.mL-1 :

The petri dishes were incubated overnight at 37°C. The number of colonies on each petri dish was then counted.

Interlab team

Our Interlab team was composed by our student intern : Camille Monteil (camille.monteil.cm@gmail.com) and Paul Ahavi (paul.ahavi@neuf.fr), which were both involved in all steps of this study.

Camille : “I think the InterLab study is a excellent practice, especially as an intern. Protocols are clear, with a lot of details, easy to understand and not such difficult to realize. Moreover Excel files, are a such a good idea and a good help.

Thanks to the InterLab study, my self-confident has definitely increased concerning experiments, and repetitive steps were maybe a little long on a day but in the end it allowed me to assimilate the manipulation and the good practices. Doing the measurements and the colony forming unit protocol on the same day was however a little stressful and difficult, maybe it's not worse in a way.”

Paul : “To me, the InterLab study was a user friendly experiment. It is perfectly suitable with the iGEM adventure because it is designed to be accessible : not too expensive and not time consuming. The main lesson I learnt from it is the way to design protocols that enable to obtain robust results. While I was impressed by your efforts to make the protocol and the explanations as clear as possible, I think the method used to process and analyse data should be more explained.

Besides, it allows me to learn how to use a plate reader, a tool that I never used before and that I needed to use in the framework of our project.”