DESIGN
Design overview
Faced with the special challenge of in situ long-term microbial-based heavy metal bioremediation, we came up with the solution of utilizing a ‘scapegoat’ – outer membrane vesicle (OMV), just like how the Monkey King did to beat the Celestial Army. By altering the functional body from the outer membrane to the vesicles it forms, we try to keep the bio-absorption of heavy metals lasting in spite of bacteria life cycle.
In an ordinary bacterial life cycle for engineered affinity towards heavy metal, the lysed cell would continue to release the heavy metal ions from the first round, forbidding any long-term attempts; In our idea, however, the heavy-metal-binding proteins just slipped from the cycle to secreted, stable OMVs. This led us to think again of the Great Sage, who rid himself of eternal recurrence by simply deleting his name from the Book of Life, Again, we mock the King of Monkeys to make an adventurous choice.
We mainly developed three parts to create a self-supporting system, integrated in a specially designed hardware device.
i. Heavy metal binding part: Creating OMV 'scapegoats'
To create the ‘scapegoat’, we need to find a reliable scaffold that can ensure the metal-binding proteins to locate and work properly on the outer membranes. A 34 kDa protein, ClyA, that is enriched on the OMVs and structurally stretched outwards, came into our sight. Frequently used as a scaffold in OMV-based antigen display, this protein can be a promising carrier of our protein.
We chose PbrR as an example heavy metal binding protein, which has a special affinity toward Pb(II).Lead contamination has been one of the most significant heavy metal contamination issue worldwide since 1970s. This formed our first part, ClyA-PbrR(BBa_K257002), which is expected to give the engineered OMVs a higher Pb(II)-binding capability.
ii. Ni-binding part: His-tag-based OMV recovery
The recovery is facilitated by His-tag, one of the most entrusted and convenient protein tag, especially useful here for the small size and the unique feature of binding affinity towards materials like zinc and nickel. It is also reported that simply displaying His-Tag on the outer membrane surface with Lpp’OmpA, a regular outer membrane display scaffold, would enable Ni-based OMV recovery. We decided it would be better if we could combine this rather small part with other parts. We briefly verified the idea with a modified version of a reported construct, Lpp’OmpA-His10(BBa_K4760004), in which we linked 10 histidine right after the common outer membrane localization scaffold, Lpp’OmpA; Meanwhile, we integrated the His-tag with the former part to create a bifunction part, ClyA-PbrR-His6(BBa_K4760000), which is later optimized into ClyA-PbrR-GGGGS-His10(BBa_K4760003).(see Engineering)
iii. OMV cross-linking to improve recovery rate
Imagine a tiny boat about to fall from the middle of Niagara Fall, and by the shore there was a group of lifeguard trying to lasso the boat with ropes – would it hang on to the shore in time? This is the vision that came to us in the first discussion after we decided using a nickel web in the hardware to recover the OMVs. Compared to the nanometer scale vesicles, even a 1mm-diameter hole would seem like a black hole.
