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 [http://parts.igem.org/Help:Assembly/3A_Assembly 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 [http://shop2.neb-online.de/4DCGI/ezshop?action=Direktanzeige&Artikelnummer=NEBIGEM1%40&WorldNr=01&ButtonName=website&skontaktid=1055557&skontaktkey=RrbLvNPZxLRIFbyyyGOZambWfZKxFK 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
Sampling
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
|