Team:Potsdam Bioware/Project/Potsdam Standard

Potsdam Standard

Problem presentation
When using the common assembly standards to assemble 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 usage of restriction enzymes 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 an 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 that 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 were mixed and transformed into E. coli. By using the RFP expression cassette we create a ligation control system due to the fact that red fluorescent colonies have a failed vector ligation.

Experimental design
Firstly, we amplified our gene of interest (GOI) with primers which had an thiophosphate overhang. This overhang is complementary to the digested restriction sites of Apa 1 and Sph 1 in the new cloning RFP standard vector. For the PCR we used the Phusion polymerase. After the PCR, we pliced the GOI with 100 mM iodine solution in 99 % ethanol and the 0.5 M Tris-HCl cleavage buffer with pH = 9.0. For doing this, we mixed 8 µL GOI with 1 µL cleavage buffer, 0.4 µL Milli-Q water and 0.6 µL iodine solution. After that we incubated the reaction mixture 5 min at 70 °C using a thermocycler. After the plicing process, the pliced GOI was mixed with the digested backbone and ligated at room temperature for 1 h and directly transformed into E.coli.

Our Experiences
We tested different combination to prove the most efficient experimental design. Therefore, we tried four different conditions: the ligation of the pliced GOI with the unpurified digested backbone with and without ligase and the ligation of the pliced GOI with the purified digested backbone with and without ligase. After transformation, we picked only clones which have lost their red fluorescence indicating that the ligation was successful.