Team iGEM14_Imperial and SCU-China 2016 have designed and characterized parts of Vitreoscilla haemoglobin (VHb). Evidence from articles and experiment prove that VHb can successfully help the strain increase its metabolism and accelerate the growth in restricted environment (Part: BBa_K1321200). In 2018, another article suggests that VHb can work more efficiently when using the twin-arginine translocase (Tat) pathway and exporting active Vhb into the periplasm of Escherichia coli1. This phenomenon is remarkable in microaerophilic atmosphere.

TorA is a twin-arginine signal peptide from E. coli. The twin-arginine system a bacterial protein export pathway with the remarkable ability to transport folded proteins across the cytoplasmic membrane. Team iGEM17_SSTi-SZGD fused TorA signal peptide directly to the N-terminal of OPH domain, successfully enabling secretion of OPH (gene product of opdA) to the periplasm of E. coli for the development of live cell biocatalysts (Part: BBa_K2244003). Inspired by these works, we improve the previous VHb (BBa_K1321200) by adding TorA signal peptide (sequence obtained from BBa_K2244003) to N-terminal of the VHb. We believe that in this new part, TorA can guide the fused VHb into periplasm, make it work well than before.

We gain VHb directly form official Kit(BBa_K1321200) and gain TorA signal peptide by company synthesis. Then VHb and TorA signal peptides overlap to a complete coding and be infused to our modified pUC19, just after lac promoter and RBS B0034. New plasmid is transformed into strain E. coli BL21-Stbl3. We also design another Stbl3 with plasmid contains VHb but without TorA signal peptide as contrast.

We select single colony from each plate culture medium then inoculate them into 20 ml LB fluid medium containing 0.1% antibiotics (except for wild type). Culture them in a shaking table at 37℃ overnight, then use M9 fluid medium to dilute them to OD600 reach 0.01. Here, each strain is separated into two kinds of growth.

Aerobic growth was conducted using 50 ml flasks containing 10 ml of culture medium under rotary shaking at 200 rpm; microaerophilic growth was conducted using a 50 ml flask containing 100 mL of culture medium under rotary shaking at 100 rpm. Collect 200 μl bacteria solution to 96well plate each 4 hours use microplate reader to monitor its OD600.

Under aerobic condition, bacteria grow faster after adding TorA signal peptide, and the K value has improved. As for microaerophilic environment, bacteria can maintain stable growth trend for a long time after adding TorA signal peptide.

All these results suggest that our new part can make VHb work better than before and give bacteria growth advance.