Team:UNSW Australia/Parts

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.

Special Part	Part Number	Part Type	Description	Designer	Length
Basic Part	BBa_K2710000	Coding	6xHis Alpha Prefoldin	Brian Ee	456
	BBa_K2710001	Coding	6xHis Beta Prefoldin	Brian Ee	396
	BBa_K2710002	Coding	6xHis Alpha Prefoldin with Spy-Catcher	Brian Ee	822
	BBa_K2710003	Coding	6xHis Beta Prefoldin with Snoop-Catcher	Brian Ee	759
	BBa_K2710004	Coding	6xHis IaaM with Snoop-Tag	Brian Ee	1749
Improved Part	BBa_K2710005	Coding	6xHis IaaH with Spy-Tag	Brian Ee	1497