Team:IIT Kharagpur/Interlab Study
INTERLAB
Overview
The iGEM inter lab study aims at collecting data regarding fluorescence from all over the world, and comparing it to understand the deviations when the same protocol is carried out around the globe. Even if the same experiment is carried out in two different places, as far as say Boston, USA and Kharagpur, India, the results may vary vastly. Moreover, unless there is some way to standardize the units of measurement, we can never compare the two results. For these two purposes, the iGEM inter lab study becomes pivotal.
Each team is provided with the inter lab kit. This kit consists of 3 test devices, along with the positive and negative controls. The teams are called upon, to use the devices provided, and measure fluorescence with whatever method available to them. This year, iGEM has provided a standard protocol for making measurements in a plate reader, a spectrophotometer or a flow cytometer. All biobricks to be tested have been sent in plasmid form. An excel sheet has been provided, to facilitate ease of data processing.
The Constructs
The Interlab study of 2016 aims to characterise the strength of 3 constitutive promoters from the Anderson Collection by measuring the fluorescence of the GFP encoded downstream of each of the promoters. The strength of the promoters can be then compared as it is directly proportional to the amount of fluorescence.
The kit contains three test devise, along with two controls. For the three test devices, the ribosome binding site(B0034) the GFP (E0040) and the terminator (B0015) are common. What they differ in, are their promoters, which are J23101, J23106 and J23117, for Test Device 1,2 and 3 respectively.
The Positive Control (PC) consists of the constitutively expressed GFP device (I20270) and the Negative Control consists of the pTetR promoter (R0040) with no coding sequence downstream of it.
All of the five parts are in the pSB1C3 backbone, which has the chloramphenicol resistance.
Test Device 1
Test Device 2
Test Device 3
Positive Control
Negative Control
Protocols:
Resuspension of plasmid DNA
The plasmids were resuspended in 100ul of Nucleus Free water, after giving a short spin (upto 6000 rpm). A short spin was again given to ensure uniform suspension.
2. Transformation in DH5α
All the plasmids were transformed into DH5α cells, following standard protocol. The transformed cells were plated onto Chloramphenicol plates, and kept at 370C for 14 hours, before storing at 40C. The transformed colonies were screened to confirm the existence of required plasmids.
3. Overnight growth at 370C,220 rpm for 16 hours
One colony from each plate was picked up and incubated overnight at 370C, 220 rpm. The incubation was carried out in 250ml conical flasks.
4. OD 600 measurement
After the overnight incubation, OD 600 for all the cultures was taken, while taking LB+Chloramphenicol as the blank. The cultures were appropriately diluted to 0.02 OD in 10ml, and kept for over day incubation.
5. Sampling
1ml samples were taken after every one hour, and stored on ice. Samples were taken from 0 hours to 6 hours. For each sample, OD 600 along with the fluorescence measurement was done
OD 600 Calibration
Path length correction was turned off. 1ml of 1X LUDOX was taken in the cuvette, and the reading was repeated 4 times. The data was imported into the provided excel sheet. The instrument used was Biospectrometer.
FITC Fluorescence Standard Curve
The serial dilution was done for FITC as prescribed in the protocol. Excitation was done at 489 nm and the emission was scanned from 500 to 600 nm. A peak for GFP was observed at 518nm. Gain:manual was set to 100. The instrument used for this was the Fluormax 300.
Raw Fluorescence Data
Results
The following depicts the graphs observed for the samples Abs 600 data:
The results indicate that the fluorescence was maximum for Test Device 1, followed by 2 and 3. This is in accordance with the expected results for the Anderson Collection.
