Team:Potsdam Bioware/Project/Potsdam Standard

Potsdam Standard

Problem presentation
When using the commonly assembly standards to assembly different parts we noticed that these procedures are very time and material consuming and therefore difficult for long and different assemblies. Another problem is the using of restrictionenzymes and gel separation in every step of the cloning process because of different conditions and efficiencies for different enzymes and the danger of mutation after UV-exposure in the gel.

Possible solution: the Potsdam Assembly Standard
The Potsdam Assembly Standard is a modified PLICing (Phosphorothioate-based ligase-independent gene cloning) method. For this standard we designed a new cloning vector with a RFP expression cassette as insert and two new restriction enzyme recognition sites in the suffix and prefix in pSB1C3. In the prefix we added the Apa I recognition site and in the suffix the Sph I recognition site. Both enzymes causing a 3’ overhang with 4 nucleotides. For the cloning process we cut the vector with Apa I and Sph I. In this assembly standard that’s the only step where we have to use restriction enzymes.
The insert was amplified with primers which contain 4 phosphothioate nucleotides and the recognition sites for Apa I and Sph I at the 5’ end. After incubation in a iodine/ethanol solution the thiophosphates were cut out resulting a 3’ overhang which is suitable to the overhangs resulting by cutting the new assembly vector. After that the digested vector and the pliced insert where mixed and transform into E. coli. By using the RFP expression cassette we create a ligation control system because red fluorescent colonies have a failed vector ligation.

Experimental design