Part:BBa_K2940001

PelB and Bpul

The PelB leader sequence was joined to Bpul laccase to enable exportation of the enzyme into the periplasmic space. Both parts were isolated from the iGEM kit via Q5 PCR and joined together using Gibson Assembly. Not only was exporting Bpul outside of the cell vital for our overall project, it also allowed us to assess the activity of Bpul without the need for expensive and time consuming purification steps.

Usage and Biology

This biobrick was produced to be an integral part of our overall azo dye bioremediation strategy. The joining of the leader sequence PelB allows for the secretion of Bpul into the periplasmic space. PelB directs the unfolded fusion protein to the periplasm, where the peptide is cleaved by a signal peptidase. This causes the protein of interest (here Bpul) to be secreted into the periplasmic space.

Characterisation

To determine the effectiveness of the PelB leader sequence in secreting Bpul into the periplasmic and extracellular space SDS page was performed. SDS analysis shows an increase in Bpul expression in induced pellet samples when compared to uninduced pellet samples. However, no bands were observed in either uninduced or induced supernatant samples. This suggests that PelB is not transporting Bpul outside of the cell, or that Bpul is effecting the folding of Bpul.

Expression analysis of Bpul from pET28a-GG-RFP-CD inE. coli BL21 (DE3). Samples were run in a 10 % SDS-PAGE gel. US -Uninduced Supernatant. UP - Uninduced Pellet. IS - Induced Supernatant. IP - Induced Pellet. The expressed 58 kDa Bpul band is indicated by a red box. A suspected increase in Bpul was observed in induced pellet samples compared to uninduced pellet.