Team:UPF CRG Barcelona/InterLab
5 th INTERLAB STUDY
Introduction
The aim is to develop a reliable and repeatable measurement based on cell number, fluorescence, absorbance (optical density) and colony formation units (CFUs). 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 center of 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 24-25 Celsius degrees). 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 need 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_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 |
H 2 O |
|
Replicate 1 |
0,056 |
0,034 |
|
Replicate 2 |
0,056 |
0,034 |
|
Replicate 3 |
0,056 |
0,034 |
|
Replicate 4 |
0,056 |
0,034 |
|
Arith. 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
Fluorescence Standard Curve
This calibration needs to be repeated because of the low R 2 (R 2 =0,8547). But it can be caused by the plate reader since it was unable to read some of the biggest concentrations in the plate (10 uM of fluorescein).
Measurement
The measurement is still in process due to some problems with the transformation of the plasmids from the Parts Collection 2018.