Overview

All BioBrick parts were constructed from plasmids which were created by Gibson assembly of DNA gBlocks® synthesised by IDT. The relevant inserts were designed to contain the iGEM protein coding prefix and suffix and cloned into pETDuet™-1 or pRSFDuet™-1 plasmids using Gibson Assembly.

These plasmids were digested and the inserts were ligated into linearised pSB1C3 plasmid backbone, to construct BioBrick parts using 3A assembly. All parts submitted to the registry were validated by sequence verification and agarose gel electrophoresis after digestion with EcoRI and PstI. Parts were also cloned into the pET19B expression vector for experimental characterisation. The protein coding parts were expressed in Escherichia coli T7 Express cells (NEB) and purified from cell lysates with Immobilised Metal Affinity Chromatography (IMAC) columns. The self-assembling functionality of our parts was further characterised by Size Exclusion Chromatography (SEC) and SDS-PAGE. From these experiments we were able to confirm the valid functionality of our parts, as they were experimentally expressed and purified, and confirmed to self-assemble as designed.