DESCRIPTION

1. The Problem

The heavy metals are still there, like a ghost of past.

Thanks to better ecological public education, nowadays everybody knows how dangerous heavy metal is: unbiodegradable, accumulable through food web, and long-term poisonous. 60 years after China’s first regulation of environmental protection, and after more than ten years of ecological devotion, it seems such hazard of pollution can never be seen again. Take the canal towns round Shanghai – there never flowed seasonal eutrophicated bloom ever again in recent years, nor would textile mills’ wastewater dye the river. From mainstream to the patty fields, clear as a mirror. Any wastewater emitted now has the heavy metal ions removed, through any possible means. Many things can absorb and precipitate these positive ions: bone charcoal, specific negative ions, even bacteria cells, which is also naturally negatively charged.

But if you look into the unseen, the heavy metal ions are still there, flowing, fleeing.

In Shanghai, there used to be countless pollutive factories. Stricter environmental policies drove most of them away, leaving behind many heavy-metal polluted lands^1,2,3. The attempts of chemical fixation, though proven practical for soil remediation in many areas⁴, doesn’t seem to work well in canal areas like Shanghai, where the water and soil share same heavy metal background. Even after the fixation, the surrounding water body will still continuously supply the land with heavy metal ions, and the polluted land had to be left vacant, like a cursed scar of the city.

We believe that Shanghai is not the only city in urge of heavy metal remediation in water-related areas. There needs to be a solution – a way to keep in situ remediation working lastingly, to round up the escaping heavy metal ions.

And we are to address it – with a completely new bioremediation strategy.

Fig.1 Figures showing heavy metal pollution hotspots in Chongming and Minhang District, Shanghai, with high concentration of 8 kinds of toxic heavy metal.²

2. Status Quo

Surface display-based biosorbents – tackle the unseen with microbial ‘hands’

Removing the dissolved heavy metal in situ is no easy job – And this time, we turn to synthetic biology, hoping to arm ourselves with biological machines. Months of discussion led to a vision of a small floating bioreactor anchored in the stream, in which we place modified microbial biosorbents that absorb heavy metals and later conveniently remove them. A reactor reduced in size can outstrip simply throwing in plants in many ways, most obviously on the space occupation: the unrestricted growth of aquatic plants has already posed great threat to round-Shanghai areas these days.

Then, picking an appropriate microbial biosorbent is our first challenge. Remember we talked about the biological heavy metal precipitation? In many synthetic biology approaches, the negatively charged feature of bacterial outer membranes (OM) is further extended with surface display of heavy metal-binding proteins (HMBP); these proteins specifically and strongly bind to one or one group of heavy metal ions, and combined with the electrostatic effect, can tightly seize the heavy metal ions, some reaching over 900 umol/g cell dry weight⁵. Surface display of HMBPs, especially on Gram- bacteria’s outer membrane, seem to be a promising choice.

Fig.2 Illustration of HMBP surface display strategy

Yet deeper research guided us to a more profound problem. Surface display presents a high removal efficiency, but after the metal binds to the bacteria surface, the bacteria must be quickly removed from the system to avoid releasing of heavy metal ions from lysed bacteria. On the other hand, to remedy the polluted land where heavy metal continuously dissolves from soil, we need the system to work lastingly.

Faced with the dilemma, we set out for an adventurous exploration of ideas. And guess who we ran into - the Great Sage Equaling Heaven.

3. The Inspiration

Inspiration of the Monkey King: using OMVs to avoid “dirty” work.

How do you keep the microorganism absorbing heavy metals without getting their membranes dirty? The famous novel Journey to the west inspired us of a totally new approach. In the novel, Sun Wukong, known as the Monkey King, has a unique talent of creating thousands of copies of himself, each costing only one hair. The smaller copies can take the simple works for him, so he can focus on the major difficulties. One of the stories described him using these hair-made golems to destroy the Celestial Army, which earned him the title of ‘the Great Sage Equaling Heaven’.

The Great Sage’s legend inspired us to adventurously revive this plot on our engineered bacteria: if we use the copies of the OM rather than itself, we could produce heavy-metal binding structure without getting the chassis affected by heavy metals.

Does such ‘scapegoat’ exist? Sure! Outer membrane vesicles, or OMVs, are 20 ~ 300 nm vesicles naturally secreted from the Gram- bacteria outer membrane, carrying the OM contents⁶. Theoretically, the OM-displayed protein should keep functioning on the OMVs as well.

Fig.3 OMV formation of Gram- Bacteria.

We instantly started to dig deeper on the idea. Multiple expert we interviewed (see Integrated-Human-Practice) supported our idea, and offered us with treasurable advice. We also came up with another candidate idea of hooking the HM-binding proteins on the inner side of OMVs, which unfortunately was declined by the calculation in Modelling. Now we stick to the idea of OMV surface display, and exploited an example construct of ClyA-PbrR-His to verify our hypothesis(see Design). Just like the Sage makes his copy golems with the hair, we can also make our engineered bacteria secrete HMBP-displaying OMVs that would then independently bind to the heavy metals and be recovered.

Fig.4 Illustration of HMBP surface display strategy

4. The Solution

Synthetic biology approaches combined with a hardware to constantly remove heavy metals.

You may wonder: how would these floating, dissolved vesicles be recovered? Would that be only another problem just as to capture the dissolved heavy metal ions? - It won’t. Synthetic biology empowered us of creating a multifunctional OMV, on which we can add much more features beyond metal-binding. What about a His-Tag? Surprisingly, we find related reports to successfully test such idea to use His-Tag interaction with nickel to recover the OMVs. Therefore, with a nickel web, we can theoretically capture all the secreted OMVs and remove the heavy metal incredibly efficiently – you just need to pull out the web.

Our adventure didn’t just stop here. A question was aroused in our minds: would a 100-nm vesicle just crash into the nickel and gets caught, when the nickel web has so apparent 1 mm or so holes (we had to led the water flow through)? This led to another innovation: we attempt to make the OMVs aggregate. Powered by one of the most entrusted part for protein-protein interaction, the SpyTag/SpyCatcher pair, we assume the OMVs can aggregate and become easier to capture. Therefore, the recovery rate of the OMVs is expected to greatly raise. In our project, we utilized a rationally designed and efficiency improved mutant pair, SpyTag003 & SpyCatcher003.⁷

Fig.5 Scheme of our final OMV product, simultaneously possessing HM-binding, Ni-binding and Crosslinking activity.

Finally, we designed a hardware device to integrate all of these functions together. This device, after tons of correction and addition of new function, can now cover every need from keeping our E.coli. chassis working smoothly to guide the water to mix with OMVs and flow through the nickel web. It’s expected to be working efficiently for weeks before we replace the nickel web and stored E.coli. cell with new ones. This hardware stands for the essence of our adventure: a multifunctional, modern attempt to create a REALLY working biological machine, to address problems in situ.

Fig.6 Two major versions of our hardware device.

Our adventure didn’t end here- it never ends. See more about our engineering cycles in the Design and Engineering pages, in which we learned, verified and improved our parts in coordination with Hardware design; or read more of our efforts in Human Practice, and Modelling to make our environment cleaner. We hope this Journey to the Heavy Metal Removal to heal not just the polluted land but also the world, by showing the world the environmental remediation potential within synthetic biology.

References

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        [3].Yuan D, He Q, et al. The heavy metal content and ecological risk waring assessment of vegetable soils in Songjiang District of Shanghai[J]. Acta Agriculturae Shanghai,2013,29(04):42-46.
        [4].Zamora-Ledezma, C. et al. Heavy metal water pollution: A fresh look about hazards, novel and conventional remediation methods. Environmental Technology & Innovation 22, 101504 (2021).
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        [6]. Sartorio MG, Pardue EJ, et al. Bacterial Outer Membrane Vesicles: From Discovery to Applications. Annu Rev Microbiol 75, 609-630 (2021).
      [7]. Keeble AH, Turkki P, et al. Approaching infinite affinity through engineering of peptide-protein interaction. Proc Natl Acad Sci U S A. 2019;116(52):26523-26533.
     [8]. Alves NJ, Turner KB, et al. Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant. Res Microbiol. 2017;168(2):139-146.