Difference between revisions of "Team:UPF CRG Barcelona/InterLab"

INTERLAB

Introduction

The aim of the Interlab Study is to develop a reliable and repeatable measurement based on cell number, fluorescence, absorbance (optical density) and colony formation units (CFUs). Each year, iGEM teams collaborate in measuring these parameters following the same protocol to obtain a way to have accurate and reliable measurements, which are essential for all sciences, including synthetic biology.

The main part of the Interlab has been always the green fluorescent protein (GFP), one of the biological markers most used in synthetic biology.

The goal of the fifth edition of Interlab is to discover the sources of variability in measurements and be able to correct them, so the measurements taken in different labs will not be variable anymore.

This year question is: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or CFUs instead of OD?

Materials and Methods

Before the starting the experimental part, a plate reader was needed. Due to the lack of plate reader in our laboratory, it was kindly asked to Proteomics and Protein Chemistry Unit (DCEXS-UPF, PRBB) to use their equipment. The plate reader is the Synergy HTX Multimode Reader from Biotek, it can measure both absorbance and fluorescence. It has pathlength correction, which was not disconnected. It has control over temperature and it was set as room temperature (around 24-25ºC). The excitation filter was 485/20 nm and the emission filter 528/20 nm and bottom optics were used. Moreover, the plates were black and flat-bottomed.

Eight plasmids needed to be characterized in DH5-alpha E.coli strain in order to follow the protocol. The strain was obtained by collaboration with BIO-IQS iGEM team. The plasmids are the following: BBa_R0040, BBa_R0040, BBa_I20270, BBa_J3604000, BBA_J364001, BBa_J364002, BBa_J364007, BBa_J364008, BBa_J364009.

The materials used over the protocol are the same ones specified in the iGEM 2018 Interlab Study Protocol.

Results

Calibration

OD600 Reference Point

	Ludox CL-X	H2O
Replicate 1	0.056	0.034
Replicate 2	0.056	0.034
Replicate 3	0.056	0.034
Replicate 4	0.056	0.034
Arithmetic Mean	0.056	0.034
Corrected Abs600	0.022	-
Reference OD600	0.063	-
OD600/Abs600	2.864	-

Table 1 | Date from the OD600 Reference Point in calibration 1.

Particle Standard Curve

Figure 1 | Graph showing the Standard curve for the calibration of OD600nm with silica beads.

Figure 2 | Graph showing the Logaritmic Standard curve for the calibration of OD600nm with silica beads.

Number of Particles	2.35e8	1.18e8	5.88e7	2.94e7	1.47e7	7.35e6	3.68e6	1.84e6	9.19e5	4.60e5	2.30e5
Mean particles / Abs600	7.15e8	6.47e8	5.04e8	4.44e8	3.82e8	3.27e8	2.94e8	2.94e8	2.04e8	1.41e8	8.36e7
Mean of med-high levels	-	4.61e8	-	-	-	-	-	-	-	-	-

Table 2 | Particles per Optical density measured.

Fluorescein Standard Curve

This calibration has a R²=0,8547.

Figure 3 | Fluorescence standard curve to calibrate the measurements with fluorescein.

Figure 4 | Logaritmic Fluorescence Standard curve to calibrate the measurements with fluorescein.

Fluorescein uM	10.00	5.00	2.50	1.25	0.625	0.3125	0.15625	0.078125	0.0390625	0.0295313	0.0097656
uM Fluorescein / a.u.	1.31E-04	6,57E-05	6.18E-05	6.00E-05	5.91E-05	5.84E-05	5.81E-05	7.17E-05	5.01E-05	4.81E-05	4.85E-05
Mean uM fluorescein / a.u.	-	6.10E-05	-	-	-	-	-	-	-	-	-
MEFL / a.u.	-	3.67E+08	-	-	-	-	-	-	-	-	-

Table 3 | Fluorescence in arbitrary units (a.u.) represented by Molecules of Equivalent FLuorescein (MEFL).

Measurement

Fluorescence per optical density

Figure 5 | Fluorescein (uM) related to OD600nm to extrapolate measures of fluorescence with optical density measurements.

Figure 6 | Fluorescence per particle measured at 0 hours and at 6 hours after growing at 37ºC

Colony Forming Units per OD600

Figure 7 | Colony forming units count on each dilution and on each device (postive and negative control)