Interlab Study

Summary

Results

Discussion

Materials and Methods

Constructs and strains

All constructs used were transformed into NEB 10 Beta cells. The constructs I20260 as well as B0015 were taken directly from the iGEM 2014 distribution plates and transformed directly. The constructs J23101.E0240 as well as J23115.E0240 were made using the 3A Assembly. Therefore the subparts J23101, J23115 as well as E0240 were transformed directly from the 2014 distribution plates into NEB 10 Beta cells. Afterwards the plasmids were recovered using the illustra plasmidPrep Mini Spin Kit. Afterwards the purified plasmids J23101 and J23115 were cut with the restriction enzymes EcoRI and SpeI, while E0240 was cut with XbaI and PstI. The restricted plasmids were then ligated together using the T4 DNA Ligase. Afterwards the Ligation product was ligated into the pSB1C3 linearized backbone provided by iGEM headquarters with the 2014 distribution after being cut with EcoRI and PstI (All restrictions and ligations were performed using enzymes and buffer of the NEB iGEM Kit). The final product was once again transformed into NEB 10 Beta cells.

The sequences of the resulting construct were confirmed by sequencing.

Note: We used the mutated version of J23115 as sent out by the iGEM headquarters. The mutation makes J23115 effectively the same promoter as K823012. We will still refer to the Promoter as J23115 though to keep it more easily recognizable with the other Interlab study results.

Inoculation and Cultivation

The cultivation of our cultures was performed in 50 ml LB Media in 500 ml shake flasks at 37 degrees Celsius and 300rpm shaking frequency. Appropiate antibiotics were added to each media (Kanamycin for I20260, Chloramphenicol for B0015, J23101.E0240 and J23115.E0240). Both antibiotics were added from a 1000* stock stored at -20 degrees Celsius for a final concentration of 35µg/ml Chloramphenicol and 50µg/ml for Kanamycin respectively.

The precultures were inoculated from the same cryo stock every time under a sterile clean bench. They were cultivated for 16 hours and then sampled for OD measurement with a Spektrophotometer. Then 2 ml of each preculture were centrifuged (5 minutes, 6000 g) and then washed twice with PBS buffer. Afterwards all cultures were inoculated to have the same starting OD once again done under sterile conditions inside a clean bench.

Sampling

To extract samples from the shake flask, these were taken out of the 37 degrees Celsius room and brought onto a nearby bench.

Measurement of OD using a Spektrophotometer

Measurement of fluorescence using a Platereader

Measurement of OD and fluorescence using the combined OD/F device of the iGEM Team Aachen 2014