In 2014 the first edition of Interlab study was launched. All iGEM teams were invited to take part in it by measuring the fluorescence of different devices. The aim of the study is "to quantify and evaluate the expression of these devices in absolute units." In other words, it aims at assessing the variation in the measurements from different labs around the world. This year, LUDOX-S30 was used as a single reference point to obtain a conversion factor to transform the absorbance data obtained to OD600 measurement. This year, there are 5 devices. The 3 test devices consist of 3 different promoters, followed by a GFP generator and ribosome binding sequences (RBS) and the other 2 devices acted as positive and negative control.
For the experiment, we followed the protocol available in the iGEM website. The protocol used was the one posted in June before the official protocol was published. The devices from Interlab Study kit were transformed into DH5-α Escherichia coli and, cultured on LB plates with correct antibiotics. The cells were incubated overnight for 14-16 hrs at 37C. Although, this year the devices were already assembled for us. We had little to no content in Device 2 and 3 tubes. When transformed Device 3 gave no colonies. Therefore, we had to clone it in the lab.
Assembly of device 3
The device was prepared by Biobrick assembly using the parts indicated in the table below. The plasmid was digested with enzymes XbaI and SpeI. The GFP fragment, E0240 was then ligated into the plasmid containing the promoter, J23117. Once ligated, the plasmid containing device 3 was transformed into DH5-α Escherichia coli. It was cultured on LB plates with antibiotic, chloramphenicol. The transformed cells were incubated overnight for 14-16 hrs at 37ºC.
Variation from protocol
In the cell measurement protocol;
1. We took 120ul sample of the cultures at each hours (0h to 6h).
2. Optical density (OD) setting wasn’t optimised on our plate reader hence, Abs600 was used for pre-dilution measurement .
Provenance and Releases
When was the work carried out for the study?
The devices were constructed and the subsequent measurements were done in the month of June. Data was analysed shortly afterwards. However, as the results were not very accurate, we repeated the experiment several times throughout the summer (especially in August.)
What is the model of measuring tool and how is it configured?
The devices were measured using a plate reader. The plate reader used was the Tecan infinite 200 with tecan-i-control software. The plate reader was used to get the absorbance at 600nm and subsequently the fluorescent read-out was got by exciting the cells with a wavelength of 490 nm and capturing the emitted wavelength with a channel of 525nm. The instrument uses a xenon-flash lamp as a light source.
The LUDOX and water replicates had OD600 values 10 fold larger than that of shown in the Interlab Study protocol. The average OD600 values for LUDOX and water replicates were 0.2801 and 0.2446, respectively. In an effort to obtain the correct OD600 figures, we attempted to troubleshoot by changing the wavelength to 400nm to 590nm. Alternating the wavelength presented a negligible difference from prior results using 600nm. This suggests that error is intrinsic in the plate reader we utilised. The correction factor calculated at OD600 was 0.4164 using reference OD600 of 0.01475. The FITC experiment was carried out to correct the cell measurement results to an equivalent fluorescein (fluorescence tracker) concentration. Our results showed at 125µM concentration of FITC, there was a significant variation between the replicates resulting in an outlier. This was mainly due to pipetting error as replicate 1, 3 and 4 were around 20,000 RFU, while replicate 2 RFU of approximately 5,000. The results of the cell measurement experiment showed that Device 2 was stronger than device 1. This result is not consistent with what we expected of the devices, as Device 1 has the strongest GFP promoter i.e., BBa_J23101. This could have been due to contamination. Device 3 had a weaker promoter to the other two devices, explaining the disparity in results. It was constructed in the lab using the part E0240 which as per the registry of standard biological parts, has been proven to have an inconsistent sequence as well.