Team:Ruia-Mumbai/InterLab
OBJECTIVE
Reliable and repeatable measurement is a key component to all engineering disciplines. The same holds true for synthetic biology, which has also been called engineering biology. However, the ability to repeat measurements in different labs has been difficult.
The goal of the fifth International InterLab Measurement Study is to identify and correct the sources of systematic variability in synthetic biology measurements, so that eventually, measurements that are taken in different labs will be no more variable than measurements taken within the same lab.The previous Interlab studies showed that by measuring GFP expression in absolute fluorescence units calibrated against a known concentration of fluorescent molecule ,can greatly reduce the variability in measurements between labs. However, when taken bulk measurements of a population of cells (such as with a plate reader), there is still a large source of variability in these measurements: the number of cells in the sample.
This is because the fluorescence value measured by a plate reader is an aggregate measurement of an entire population of cells, we need to divide the total fluorescence by the number of cells in order to determine the mean expression level of GFP per cell. Usually it is done by measuring the absorbance of light at 600nm, from which we compute the “optical density (OD)” of the sample as an approximation of the number of cells. OD measurements are subject to high variability between labs, however, and it is unclear how good of an approximation an OD measurement actually is. Hence this year’s Interlab study mainly focused on standardizing GFP expression for each cell. The absorbance and fluorescence of the cell populations were used to calculate cell concentration by Calibration Curve method. Moreover, absolute cell number of the samples was obtained by performing Standard plate count.
DEVICES
PLATE READER:
INTERLAB DATA ANALYSIS
We at Team Ruia-Mumbai tried our hands at working for an international collaborative experiment of publishable quality by participating in the IGEM Fifth International Interlab Measurement Study. It is a great feeling to be part of a world-wide community working for standardizing measurements in synthetic biology. We realized the importance of planning, precision and accuracy while performing experiments and were amazed by the variabilities caused due to so minute changes. We learned team spirit and work ethics. Moreover, we experienced the sheer joy and satisfaction when our Interlab data got accepted in first attempt!!
This year’s Interlab study mainly focused on standardizing GFP expression for each cell. The absorbance and fluorescence of the cell populations were used to calculate cell concentration by Calibration Curve method. Moreover, absolute cell number of the samples was obtained by performing Standard plate count. The above two approaches were used to determine the exact fluorescence value for each cell. We performed both plate reader as well as Flow cytometry protocol as a part of the IGEM Interlab study 2018. We had a fruitful collaboration with National Institute for Research in Reproductive Health (NIRRH), Mumbai for the Interlab study as we don’t have access to 96-well plate reader and flow cytometer.
Data is just information till we analyse it. Once analysed, it becomes a powerful tool for scientific study. We analyzed our Interlab data and tried to interpret it in best possible ways. The Excel sheets provided by IGEM measurement committee 2018 were noteworthily explicit explaining all the calculations precisely. The sheets helped us immensely in our Interlab data analysis.
LUDOX CALIBRATION
Low absorbance values were observed as expected for both LUDOX (~0.07) as well as ddH2O (~0.03).
MICROSPHERE CALIBRATION
- Microsphere beads settled in less than 10 mins (~10 secs)
- The values obtained did not give a perfect 1:1 plot as expected for both the graphs. However the calculated R2 values were approximately equal to 1.
- The microsphere calibration protocol gives us a standard plot of Absorbance (A600) vs Particle size, which is equivalent to the size of E.coli cells (DH5 alpha). The values obtained in the cell measurement protocol, can be extrapolated on this curve to get the corresponding cell number.
For ex: If we get values of absorbance 600 of a cell sample as 0.5 Abs units.
Following calculations can be performed to determine the cell no. of the sample.
Y=mx+c
y = 3E-09x + 0.0392
0.5 = 3E-09 * cell number + 0.0392
Cell number = 0.5 – 0.0392 / 3E-09
Therefore cell number = 1.536 * 10^8 cfu/100 uL
- We obtained almost constant Abs600 values for higher dilutions which we suspect is because of:
- Pipetting errors
- Microspheres at higher dilutions might be in negligible concentration causing their Abs600 values to tend to the values of blank. Abs600 values of the 4th replicate were observed to be low compared to the previous replicates which may be due to the settling of microsphere between the reading frames of the instrument.
FLUORESCEIN CALIBRATION
Fluorescein calibration protocol passed all the common sense tests i.e. the fluorescence readings reduced 1/2 times with every dilution and the blank showed less fluorescence than the test.
The fluorescence readings gave an almost perfect 1:1 plot on both linear and log scale. The drifting of higher dilution readings from the trendline may be attributed to the pipetting error.
The fluorescence readings of the test devices could be extrapolated on the fluorescein calibration curve obtained by this protocol using the above equations to get an estimate cell number of the respective test devices.
CELL MEASUREMENT PROTOCOL
The competent E.coli DH5α cells were transformed with 8 devices provided in the iGEM distribution kit. The transformed cultures were viewed under fluorescence microscope as 7 out of 8 devices express GFP.
The 1:10 diluted overnight culture was used for setting the target Abs600 to 0.02 in 12 ml. However the 0 hour OD600 and fluorescence readings were taken after an hour delay due to unavailability of instrument at 0 hour. Till then the 0 hour samples were kept on ice. Nonetheless, the 0 hours samples showed a threefold increase in their OD600 readings.
The 1:10 diluted Abs600 measurement of one of the two biological replicates of devices 2F and 2P was found to be too low compared to their counterpart. Hence, the subsequent mathematical calculations expressed the need of adding more than 5 ml of 1:10 diluted culture to set the target Abs600. As a result, the Abs600 of the above two colonies was set to 0.02 units using the overnight culture.
The net fluorescence in a.u.values increased for all 7 devices excluding negative control after 6 hours. However, the fluorescence per OD and fluorescence per particle values for 6 hours for all the devices decreased when compared to their O hour readings inspite of increase in Abs600 unlike the proportionate increase in fluorescence compared to increase in Abs600 as expected. We hypothesize that it might be happening because all the increased cell population might not be fluorescing to the same extent.
