Difference between revisions of "Team:UNSW Australia/Parts"

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<h2>Overview<h2>
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<h2>Overview</h2>
 
<p>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.</p>
 
<p>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.</p>
 
<p>These plasmids were digested and ligated with the linearised pSB1C3 plasmid backbone provided by iGEM, to construct BioBrick parts using 3A assembly. All parts submitted to the registry were validated by sequence verification and a diagnostic enzyme digest gel. 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.</p>
 
<p>These plasmids were digested and ligated with the linearised pSB1C3 plasmid backbone provided by iGEM, to construct BioBrick parts using 3A assembly. All parts submitted to the registry were validated by sequence verification and a diagnostic enzyme digest gel. 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.</p>

Revision as of 04:06, 17 October 2018


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 ligated with the linearised pSB1C3 plasmid backbone provided by iGEM, to construct BioBrick parts using 3A assembly. All parts submitted to the registry were validated by sequence verification and a diagnostic enzyme digest gel. 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