Interlab

1. OBJECTIVE

Accurate, precise and reproducible measurement is a key component of all disciplines and synthetic biology is not an exception for that. However, the ability to reproduce measurements in different labs is a difficult task to achieve.
The goal of the Fifth International Interlab study was standardizing GFP expression per cell across all the world laboratories. GFP is widely used as a measurement tool in synthetic biology. Hence, it is of immense importance to standardize the GFP expression for all laboratories so that they can compare and build upon each other’s data. Last year’s iGEM Interlab study focused on standardizing GFP expression for a cell population calibrated against a known concentration of fluorescent molecule. This year, the study has been extrapolated to calculate GFP expression for a single cell thus eliminating error caused due to cell-to-cell variability. Moreover, this study has been carried out by laboratories across the world using a standardized protocol provided by iGEM Measurement Committee to minimize lab-to-lab variability.

For the Fifth International Interlab study 2018, 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 at zero hour was obtained by performing Standard plate count. The above two approaches were used to determine the exact fluorescence value per cell.

We, Team Ruia-Mumbai performed both the plate reader as well as the 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.

2. EXPERIENCE

We, Team Ruia-Mumbai tried our hands on working for an international collaborative experiment of publishable quality by participating in the IGEM Fifth International Interlab Measurement Study 2018. It is a great feeling to be part of a world-wide community working for standardizing measurements in synthetic biology. After going through the protocol for plate reader and flow cytometer for around 10-15 times, the next challenge for us was of executing the experiments.The execution of experiment required three to four days of rigorous planning and there we realized the importance of planning and patience. We got an opportunity to analyse our data using a flow cytometer in collaboration with NIRRH and it was a great experience of learning new techniques. The Excel sheets provided by IGEM measurement committee 2018 were explicit explaining all the calculations precisely. The sheets helped us immensely in our Interlab data analysis.Data is just information till we analyse it. Once analysed, it becomes a powerful tool for scientific study.

While analysing the data from the plate reader and the flow cytometer we were amazed by the readouts and trends within biological and technical replicates. We learnt team spirit and work ethics. Moreover, we experienced the sheer joy and satisfaction when our Interlab data got accepted in the very first attempt!!

4. PLATE READER

LUDOX CALIBRATION

1. Low absorbance values were observed as expected for both LUDOX (~0.07) as well as ddH2O(~0.03).

MICROSPHERE CALIBRATION

1. Microsphere beads settled in less than 10 mins (~10 secs)
2. 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.
3. 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
 ∴cell number = 1.536 * 10^8 cfu/100 uL


4. 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.
5. 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

1. 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.
2. INCOMPLETE CONTENT
3. 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.








4. The test device 3, 5 and 6 are the devices showing maximum fluorescence. The relative order of fluorescence shown by these three devices is as follows:-
   
   



5. We always faced a time lag between setting up OD600 of starting sample and taking measurements after pipetting on 96 well plate reader due to the sheer enormity of pipetting such a huge number of samples.

CFU COUNT PROTOCOL

1. The overnight culture was diluted and the OD600 was measured. The culture was set to 0.1OD600 after doing relevant mathematical calculations using the diluted culture. Subsequently the OD600 measurements were taken after a delay of one hour due to machine availability by which time the readings obtained were approximately 0.2OD600. Hence, all the 0.2OD cultures were 1:2 diluted to get a projected OD600 of 0.1. The OD600 readings after 1:2 dilutions were not taken due to unavailability of the plate reader at the required time.
2. We found it difficult to prepare 1:20 dilutions in 2ml eppendorrf tubes as the solution was filled up to the brim making it difficult to aliquot for further dilutions and effective vortexing.
3. The results obtained for cfu count were as follows:-




All the replicates of both positive and negative control devices showed Too numerous to count (TNTC) colonies in lowest dilution plated and too less colonies in highest dilution plated. In the dilution having countable number of colonies, the replicates did not show much uniformity.



Legend:-
Series 1 – Colony 1
Series 2 – Colony 2

The cfu count/ml for 0.1OD600 cultures did not show much congruency between the two biological replicates – colony 1 and colony 2 for both positive and negative control devices.

However, we got an estimate cfu count for both the biological replicates of positive and negative control devices as follows:-