InterLab

Studying Fluorescence

The goal of this year’s InterLab Study was to identify and minimize the sources of systematic variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of optical density (OD).

Participating in the fifth iGEM InterLab Study was a great opportunity to start this year’s competition as well as acquire some valuable knowledge which we implemented into practice during the project.

Description

At the beginning of the InterLab study we completed three distinct calibration protocols. At first, we performed the LUDOX Protocol in order to obtain a conversion factor to transform absorbance (Abs600) from the plate reader into a comparable OD600 measurement as would be obtained with a spectrophotometer. Next, we completed the Microsphere Protocol as it allows a standard curve of particle concentration which is used to convert Abs600 measurements to an estimated number of cells. Finally, by completing the Fluorescein Protocol we generated a standard fluorescence curve which is used to compare fluorescence output of different test devices. Completion of the calibrations ensured that we take cell measurements under the same conditions. It is worth mentioning that prior calibration, we prepared competent E. coli DH5-alpha cells and transformed them according to the standard transformation protocol. During all of the experiments we tested 8 plasmids: 2 controls and 6 test devices (Tab 1).

Tab. 1 Parts received and tested during iGEM’s fifth InterLab Study

Device	Part Number	Features
Negative control	BBa_R0040	Medium strength promoter, promoter is constitutive and repressed by TetR
Positive Control	BBa_I20270	J23151 inserted in the Promoter MeasKit
Test Device 1	BBa_J364000	GFP expressing constitutive device
Test Device 2	BBa_J364001	GFP expressing constitutive device
Test Device 3	BBa_J364002	GFP expressing constitutive device
Test Device 4	BBa_J364007	Expresses GFP under the control of a constitutive promoter from the Anderson collection
Test Device 5	BBa_J364008	Expresses GFP under the control of a constitutive promoter from the Anderson collection
Test Device 6	BBa_J364009	Expresses GFP under the control of a constitutive promoter from the Anderson collection

RESULTS AND DISCUSSION

1. MEASUREMENT OF LUDOX CL-X OD600 REFERENCE POINT

Using LUDOX CL-X as a single point reference allowed us to obtain a ratiometric conversion factor to transform absorbance data into a standard OD600 measurement. This is crucial to ensure that plate reader measurements are not volume dependent. After this calibration part we obtained a radiometric conversion factor (Tab. 2) which will be used in further Interlab study measurements.

Tab. 2 LUDOX CL-X measurement. Obtained ratiometric conversion factor is 3,419

	LUDOX CL-X	H2O
Negative control	BBa_R0040	Medium strength promoter, promoter is constitutive and repressed by TetR
Positive Control	BBa_I20270	J23151 inserted in the Promoter MeasKit
Test Device 1	BBa_J364000	GFP expressing constitutive device
Test Device 2	BBa_J364001	GFP expressing constitutive device
Test Device 3	BBa_J364002	GFP expressing constitutive device
Test Device 4	BBa_J364007	Expresses GFP under the control of a constitutive promoter from the Anderson collection
Test Device 5	BBa_J364008	Expresses GFP under the control of a constitutive promoter from the Anderson collection
Test Device 6	BBa_J364009	Expresses GFP under the control of a constitutive promoter from the Anderson collection

2. GRAPHING A SILICA MICROSPHERE ABSORBANCE (Abs600) STANDARD CURVE

Monodisperse silica microspheres exhibit size and optical characteristics similar to cells, with the additional benefit that the number of particles in a solution is known. Therefore, this measurement allowed us to construct a standard curve which can be used to convert Abs600 measurements to an estimated number of cells.

Fig. 1

Fig. 1 LUDOX CL-X measurement. Obtained ratiometric conversion factor is 3,419.

Fig. 2

Fig. 2 Particle standard curve generated by measuring the absorbance of serial dilutions of silica microspheres (known amount of particles per volume) displayed in a log scale to demonstrate a linear relationship between particle count per volume and absorbance.

During this calibration part we obtained two particle standard curves which are important for proper cell measurement. However, we can observe a curve in the log scale graph (Fig. 1), although it should have a 1:1 slope. We assume that this inconsistency could have been due to pipetting errors or an oversaturated detector.

3. GRAPHING A FLUORESCEIN FLUORESCENCE STANDARD CURVE

In the last part of the calibration we prepared a dilution series of fluorescein in four replicates and measured the fluorescence. During this calibration part we generated a standard curve of fluorescence for fluorescein concentration.

Fig. 3

Fig. 3 Standard curve of fluorescein generated by measuring the fluorescence of serial dilution stock (µM). Fluorescence is plotted against the fluorescein concentration.

Fig. 4

Fig. 4 A standard curve of fluorescein generated by measuring the fluorescence of serial dilution stock (uM). Fluorescence is plotted against the fluorescein concentration on a logarithmic scale.

During this calibration part we generated a standard curve of fluorescein. Standard curves (linear and on a logarithmic scale) have a 1:1 slope which ensures us that there were no significant mistakes during this calibration part and the data can be used for cell measurement. This allows us to successfully convert cell based readings to an equivalent fluorescein concentration.

CELL MEASUREMENTS

For cell measurements we used the same settings that we used in our calibration measurements. At first, according to the standard protocol we transformed cells with 8 different plasmids (Tab. 1). We picked 2 colonies from each transformation plates and inoculated in 5-10 mL LB medium + Chloramphenicol. We grew the cells overnight (16-18 hours) at 37 °C and 220 rpm. After that we diluted the cultures to a target Abs600 of 0.02. We took samples from these diluted cultures prior to incubation and after 6 hours of incubation measured Abs600 (Fig. 5) and fluorescence (Fig. 6).

Fig. 5

Fig. 5 Graph comparing the raw Abs600 prior incubation and at hour 6 for each colony using each control/device

Fig. 6

Fig. 6 Graph comparing the raw fluorescence prior to incubation and at hour 6 for each colony using each control/device

Comparing absorbance and fluorescence of cells prior to incubation and after 6 hours we can observe that absorbance as well as fluorescence were more intense after 6 h of incubation as it was expected. Based on the assumption that one bacterial cell gives rise to one colony, colony forming units per 1 mL of an OD600 = 0.1 culture was calculated by counting the colonies on each plate with fewer than 300 colonies and multiplying the colony count by the Final Dilution Factor on each plate The results are shown in Tab. 3.

Tab. 3 Colony forming units (CFU) per 1 mL of an OD600 = 0.1culture

Samples	CFU/ml in Starting Sample
1.1 Positive Control	0.132667	1.2 Positive Control	0.086667	1.3 Positive Control	0.271333	2.1 Positive Control	0.448667	2.2 Positive Control	0.394667	2.3 Positive Control	0.659667	3.1 Negative Control	0.236	3.2 Negative Control	0.722	3.3 Negative Contro	0.346667	4.1 Negative control	0.494	4.2 Negative control	0.279	4.3 Negative control	0.395

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Team:Vilnius-Lithuania/InterLab