Team:Unesp Brazil/InterLab
iGEM Unesp Brazil
InterLab
Introduction
Although previous studies in Interlab demonstrated the variation on measures between labs can be reduced with the GFP expression measurement in absolute units of fluorescence, there are some limitations in relation to the number of cells in the sample.
In order to solve this problem, the InterLab proposed a method to investigate the absolute count of cells or colony forming units as an option to measure cell density, in an effort to reduce the variation in the fluorescence analysis between labs.
To collaborate with the study, the iGEM Unesp Brazil team executed all the protocol procedures with the intention to send the final results to the mediation committee from iGEM.
Materials and methods
Calibration and Measurement
To start the analysis, three calibration curves were constructed following the protocols provided:
- OD 600 Reference point: Using LUDOX as a reference to convert measurement data (OD 600) into the plate reader for an equivalent obtained from a spectrophotometer.
- Particle Standard Curve: From a pattern of silica particle concentration similar to the cells, carry out the conversion of the measurements (OD 600nm) to a number of cell units.
- Fluorescence standard curve: Through a GFP-like molecule, fluorescein, to obtain a standard fluorescence curve to convert cell-based readings to an equivalent concentration of fluorescein.
Devices and controls
The positive and negative devices and controls provided in the Measurement Kit are listed below. All of the tests and controls have the plasmid pSB1C3 as a template and as requested, all were transformed into E. coli DH5α cells, following the transformation protocol provided by iGEM.
Negative control (BBa_R0040)
Positive control (BBa_I20270)
Test Device 1 (BBa_J364000)
Test Device 2 (BBa_J364001)
Test Device 3 (BBa_J364002)
Test Device 4 (BBa_J364007)
Test Device 5 (BBa_J364008)
Test Device 6 (BBa_J364009)
Plate reader: Tecan Infinite M200 PRO.
OD600 absorbance
Wavelenghts: 600nm
Number of Flashes: 10
Settle time: 0ms
Plate: Costar 96 Flat Bottom Transparent Polysterene
Fluorescence measurements
Mode: Fluorescence Top Reading
Excitation Wavelenght: 485nm
Emission Wavelenght: 535nm
Gain: 52
Number of Flashes: 10
Settle time: 0ms
Z-Position: 16.9mm
Colony Forming Units (CFU)
Based on the assumption that 1 colony is derived from 1 bacterial cell, the colony-forming unit counting procedure (CFU) lists the number of cells per mL with measured OD. To do this, we proceeded according to the protocol, taking into consideration only the plaques that presented to number of colonies smaller than 300.
Results
OD600 Reference Point
Particle Standard Curve
Fluorescein Standard Curve
Fluorescence per OD
Fluorescence per particle
Colony Forming Units per 0.1 OD E. coli cultures
Conclusions
The InterLab challenge provided a great opportunity for team members who do not work hard within the lab to have a chance to learn and develop their skills throughout the process.
In this edition, we faced problems during the transformation stage because some devices did not grow in the plate, being necessary to repeat all the methodology with a higher amount of plasmid.
When analyzing the cell density and fluorescence obtained, we reached a deadlock: Although devices 1 and 5 showed an increase in fluorescence within 6 hours, we noticed that there was a limitation in growth and, however, device 3 had a growth with an absence of fluorescence. This contrariness certainly affected our values of the fluorescence ratio by OD and particle fluorescence for these devices.
In addition, another step that generated greater difficulties was the counting of the colony-forming units, since the dilution methodology required a lot of attention and, because of this, we had to repeat it to obtain a more satisfactory final result and to be able to estimate the number of viable cells.