iGEM Dundee 2013 · ToxiMop


We have submitted two BioBricks to the Registry of Standard Biological Parts that will hopefully be of use to future teams and projects.

1. BBa_K1012001 Protein Phosphatase 1

Human Protein Phosphatase 1 (PP1) is a protein from the family of serine/threonine phosphatases, we have used it as a microcystin binding protein however it regulates many processes in the body therefore it may be used in many other ways.

2. BBa_K1012005 ompC-GFP reporter construct.

This is an improved version of BBa_R0083. BBa_R0083 comprises the ompC promoter, containing OmpR-binding sites. To improve this brick we added a strong Ribosome Binding Site (RBS; from BBa_B0034) followed by Green Fluorescent Protein (BBa_E0040). This was achieved by digesting BBa_R0083 with SpeI + PstI . The RBS from BBa_B0034 was excised with XbaI / PstI, and ligated into the BBa_R0083. The resultant plasmid was digested with SpeI + PstI , and was ligated with the GFP-encoding gene which had been excised from BBa_E0040 by digestion with XbaI / PstI. The resultant plasmid, Bba_ K1012005 responds to the osmotic activation of the EnvZ by producing green fluorescence. This part has been verified to work in this way.

In between the European and World Jamborees, we have successfully constructed the following BioBricks we ran out of time preparing for the European Jamboree.

3. BBa_K1012002 The TorA (Tat-targeting) signal sequence

This BioBrick contains DNA coding for the TorA (TMAO reductase) signal peptide. This part can be added at the N terminus of a protein of interest, thus targeting it for export across the bacterial cytoplasmic membrane by the twin arginine transport system (Tat) which transports folded proteins. We used this part to export pre-folded PP1 from the cytoplasm to the periplasm of E. coli.

4. BBa_K1012004 The MalE (Sec-targeting) signal sequence

This BioBrick contains DNA coding for the MalE signal peptide. Addition of this part to the N terminus of a desired protein will target the protein product for export by the general secretory pathway (Sec) which transports linear polypeptides. We used this part to target PP1 for export to the periplasm by the Sec machinery.