Team:Aalto-Helsinki/InterLab

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InterLab Study

This year, The iGEM Foundation invited us to participate in the Interlab Study and we agreed to take part in it. In the study, iGEM teams gather fluorescence data for three GFP expressing devices around the world using the same methods for cloning and measurements. The goal of this international experiment is to find out, how precisely organisms act the same in the different parts of the world if they contain similar genetic material. Our InterLab Study Lab Book can be found here for a fully detailed overview of our study.

Devices

The following devices were created in a backbone pSB1C3:

⚫ J23101 + I13504 (B0034-E0040-B0015)
⚫ J23106 + I13504 (B0034-E0040-B0015).

However, constructing a device J23117 + I13504 (B0034-E0040-B0015) wasn't successful due to the time limit and limited ligation times. More details available on Lab Book. The device sizes were analyzed with restriction mapping and the results can be seen in Figure 1.

Used BBa_I20270, a GFP expressing part in the pSB1C3 backbone as a positive control. Negative controls were organisms with an empty plasmid and without any vector.

Protocols

Constructing devices

Protocols for the restriction, ligation and transformation of BioBricks can be found here.

Cultivations and measurement

Followed the next protocol for preparing the samples:

Streaked out LB-plates with E.coli TOP10 strain containing every device and control with chloramphenicol concentration of 35 μg/ml. Incubated plates overnight (18-20 hours) at 37\(^\circ\)C. Picked up biological triplates from the plates and inoculated 3 ml liquid cultures from the colonies with 12 ml polypropylene test tubes. Incubated the tubes at 37\(^\circ\)C with shaking at 300rpm for 18 hours. Measured OD600 values of cultures in cuvettes and calculated the dilution required for each sample to obtain the OD value of 0.5. Diluted and re-measured the samples to reach 5% accuracy of 0.5. Transfered 200 μl of each sample into 96-well transparent plate and set the platereader to read fluorescence intensity. Proceeded with the measurements with the following equipment:

Type: Cell Imaging Multi-Mode Plate Reader
Name and model: Cytation 3
Company: BioTek Instruments, Inc

The following parametres were used:

Excitation wavelength: 470 nm
Emission wavelength: 511 nm
Optics Position: Bottom
Filter: Quadruple Monochromators with Bandwith of 16 nm
Units Reported: RFU

Results

The measurements were partially successful. Results can be seen in tables below. The data hasn't been processed further and the units are in Relative Fluorescence Units.

Most samples generated overflow values which could not be measured with the plate reader. The incubation may have generated too much fluorescent proteins even though the samples were diluted properly. The problem wasn't solved after new dilutions of 9:10 ratio and due to the time limit, other ratios weren't attempted. The first blank value is with antibiotics and the second without for the negative control without any plasmids.

1st Technical Replicon

Sample	J23117 + I13504	J23101 + I13504	J23151	Empty backbone	Without backbone	Blanks
1	OVRFLW	83954	53925	16309	13663	29336
2	OVRFLW	OVRFLW	56764	16335	14148	14940
3	OVRFLW	OVRFLW	61918	16771	14073

2nd Technical Replicon

Sample	J23117 + I13504	J23101 + I13504	J23151	Empty backbone	Without backbone	Blanks
1	OVRFLW	85224	53553	16307	13747	29470
2	OVRFLW	OVRFLW	56472	16204	14267	14972
3	OVRFLW	OVRFLW	60969	16818	14381

3rd Technical Replicon

Sample	J23117 + I13504	J23101 + I13504	J23151	Empty backbone	Without backbone	Blanks
1	OVRFLW	86750	56390	16123	13536	30778
2	OVRFLW	OVRFLW	58294	16294	14207	14856
3	OVRFLW	OVRFLW	62229	16793	13994

Validation

The gel electrophoresis mapping of J23101 + I13504 and J23106 + I13504 can be seen in Figure 1 where the outermost bands belong to ladders. Both the samples (in the middle) should have the same size (2989 bp). The bands belonging to constructs seem to be slightly bigger (~3000 bp) than expected but it is hard to say if the ladders have a bit of inaccuraty.

Figure 1. Restriction mapping for J23101 + I13504 and J23106 + I13504.