Part:BBa_K929301
Potsdam Standard Backbone
Introduction
The Potsdam Assembly Standard is a modified PLICing (Phosphorothioate-based ligase-independent gene cloning) method (Blanusa et al. (2010)). 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.
Our experiences
For culturing E.colis transformed with Potsdam Standard Cloning Vector with RFP:
The red fluorescence of the transformed colonies is obvious after 24 h incubation at 37 °C and over night incubation at 8 °C. To speed up the cloning, you can use LED light to see red fluorescence even after 16 h incubation at 37 °C. The transformed E.colis don't need any IPTG for RFP production. For the over night culture (fig. 1), we observed that it is better to incubate them with IPTG. After centrifugation, you can see the cerry-like cell pellets (fig. 3).
Experimental Design: How to clone with the Potsdam Standard
The insert is amplified with primers that contain 4 phosphothioate nuclheotides and the recognition sites for Apa I and Sph I at the 5’ end. After incubation in an iodine/ethanol solution, the thiophosphates were cut out resulting in a 3’ overhang which is suitable to the overhangs that was created by cutting the Potsdam Standard vector.
After that, the digested vector and the PLICed insert are mixed and transformed into E. coli (Summary: fig. 1). By using the RFP expression cassette (fig. 2), we created a ligation control system. Due to the fact that red fluorescent colonies have a failed vector ligation we can tell which colonies are correctly ligated. The colonies that do not show red fluorescence are the positive clones.
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