Team:Fudan-CHINA/InterLab
Interlab
"No road of flowers lead to glory."
This year, Team Fudan-CHINA is participating in the fifth iGEM interlab study to determine correlation between fluorescence protein expression, cell absorbance and cell density.
Materials:
-iGEM Measurement Kit
-test plasmids delivered in Part Distribution Kit Plates
-self-made competent E.coli DH5α strain
-Bio-Tek Cystation plate reader
-Commonly used laboratory consumables
Calibration #1 – OD600 reference
LUDOX sample was prepared according to the interlab protocol of which OD600 and Abs600 was read through plate reader to establish a transformation of absorbance raw data into OD600 measurements. Path length fixation was turned off during this and every following test.
Figure 1.1: OD600 reference
Calibration #2 – Absorbance standard curve
Serial dilution of silica beads was prepared on a 96 well plate according to the interlab protocol and Abs600 of each well was tested. An absorbance curve was generated using those data collected which seemed to partly deviate from a strict linear relationship. This should result from pipetting errors.
Figure 1.2: absorbance standard curve
Calibration#3 – fluorescence standard curve
Serial dilution of standard fluorescein was prepared according to the interlab protocol and fluorescence of each well was tested under the condition of 485nm excitation and 528nm emission. Measurement gain was tuned to 50 to keep readings under OVERFLOW and was maintained during following tests.
Figure 1.3: fluorescence standard curve
Cell measurement
Each test plasmid was transformed into E.coli DH5α and single colony obtained on the plate. 2 colonies was picked and cultivated in 5mL LB medium + Chl overnight. Samples of 0hr and 6hr of incubation was prepared according to interlab protocol. Please be noted that cultures were diluted to Abs600 0.02 in 12mL LB + Chl in the original 15mL tube where it was grown overnight instead of a new 50mL tube. Besides, Abs600 of overnight culture was read and dilution to 0.02 calculated according to equation provided in the interlab protocol but dilution results seemed to deviate from expected Abs600 of 0.02. Please refer to our submitted data for further information.
Figure 2.1: cell measurement readings
Colony Forming Units per 0.1 OD600
Cell cultures were grown and prepared according to the interlab protocol and spread on LB + Chl plates to determine CFU. Please be noted that due to lack of 2mL tube some dilutions of overnight culture were mixed in a 12 well plate and shaking was performed instead of vortexting tubes before pipetting. Besides, each culture was tested in 2 repeats instead of 3 due to shortage of appropriate Chl+ plates. Raw data were submitted in the questionnaire.
Materials:
-iGEM Measurement Kit
-test plasmids delivered in Part Distribution Kit Plates
-self-made competent E.coli DH5α strain
-Bio-Tek Cystation plate reader
-Commonly used laboratory consumables
Calibration #1 – OD600 reference
LUDOX sample was prepared according to the interlab protocol of which OD600 and Abs600 was read through plate reader to establish a transformation of absorbance raw data into OD600 measurements. Path length fixation was turned off during this and every following test.
Figure 1.1: OD600 reference
Serial dilution of silica beads was prepared on a 96 well plate according to the interlab protocol and Abs600 of each well was tested. An absorbance curve was generated using those data collected which seemed to partly deviate from a strict linear relationship. This should result from pipetting errors.
Figure 1.2: absorbance standard curve
Serial dilution of standard fluorescein was prepared according to the interlab protocol and fluorescence of each well was tested under the condition of 485nm excitation and 528nm emission. Measurement gain was tuned to 50 to keep readings under OVERFLOW and was maintained during following tests.
Figure 1.3: fluorescence standard curve
Each test plasmid was transformed into E.coli DH5α and single colony obtained on the plate. 2 colonies was picked and cultivated in 5mL LB medium + Chl overnight. Samples of 0hr and 6hr of incubation was prepared according to interlab protocol. Please be noted that cultures were diluted to Abs600 0.02 in 12mL LB + Chl in the original 15mL tube where it was grown overnight instead of a new 50mL tube. Besides, Abs600 of overnight culture was read and dilution to 0.02 calculated according to equation provided in the interlab protocol but dilution results seemed to deviate from expected Abs600 of 0.02. Please refer to our submitted data for further information.
Figure 2.1: cell measurement readings
Cell cultures were grown and prepared according to the interlab protocol and spread on LB + Chl plates to determine CFU. Please be noted that due to lack of 2mL tube some dilutions of overnight culture were mixed in a 12 well plate and shaking was performed instead of vortexting tubes before pipetting. Besides, each culture was tested in 2 repeats instead of 3 due to shortage of appropriate Chl+ plates. Raw data were submitted in the questionnaire.
iGEM 2018 interlab measurement study page: https://2018.igem.org/Measurement/InterLab
2018 interlab plate reader protocol(pdf): https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf
Address
G604, School of Life Sciences, Fudan University
2005 Songhu Road, Yangpu, Shanghai, China
2005 Songhu Road, Yangpu, Shanghai, China
