Biobricks

All our parts are listed in our wiki page Parts, where are also reported the links to the description of every part in the iGem Registry.

Here, we also show how we contributed to the characterization of pTAC, trp & lac regulated promote BBa K864400, as we utilized it to verify its induction for the surface-expression of dehalogenase DeHa2: BBa_K5109023. We strongly believe that our biological system could be widely implemented in many more bioremediation projects, featuring different enzymes.

We created a new series of simple basic parts: some of them are new basic parts meanwhile some others are improvements on already existing basic parts that we modified to adapt them for the experimental part of our project. All our new basic and composite parts are compatible with RFC 10 standard. During the process of selecting and designing our parts, we conducted a series of bioinformatic analyses to select the most promising enzymatic candidates for degrading PFAS. Specifically, we first searched for orthologous enzymes using BLAST, then we used InterProScan to analyze the protein sequences, and lastly we performed docking simulations using AutoDock VIna and CB-Dock2.
With those basic parts, we built a series of new composite parts that we used in our experiments: our purpose is to test new biological tools for bioremediation, in particular to see how different types of Laccases and Dehalogenases work to degrade PFAS. In order to do so, our new composite parts provide surface display systems for four different enzymes.

During the design of a surface expression tool, a carrier element is needed to bring a passenger to the outer membrane surface of the cell. In our case, the carrier is an Lpp-OmpT construct which acts as a membrane anchor. We made the choice of inserting the carrier’s sequence with different enzymes, this allows to extract the anchor if needed and to transform the surface display system into an intracellular expression tool, all of this without altering the RFC 10 compatibility of the composite part.

Filtering system

With this module of our project we wanted to give our contribution in the transition from the thermal treatment of saturated granular activated carbons (GAC) filters to a chemical one. This change is needed as subjecting GAC to high temperatures can change its chemical and physical properties, lowering its sorption capacity, and it could lead to the emission of volatile and mobile products and harmful compounds.
We first investigated the effectiveness of two GAC desorption solutions, obtained from a review of the literature, then we shifted our attention to another adsorbent: anion exchange resins (AER).
From our experiments, we realized that further investigation of GAC desorption solutions are necessary, bearing in mind that potentially the volume of such solutions must be low, so as to be environmentally friendly and economically feasible.
We then explored the resins and realized, both through literature and experimentally, their effectiveness as filters against PFAS.
Furthermore, to be regenerated, these adsorbents require sodium chloride-based solutions, which are not harmful to the environment. Moreover, very promising regeneration yield values ​​are reported in the literature, which is why we decided to carry out experiments aimed at reproducing the same results.

For more information visit Plant Design - Filters.

Sensor prototype

Even though the results we obtained are very preliminary, they hold great scientific value. Our decision to start from scratch with a new sensor model was quite risky. There was a chance that we could have worked on the project for a long time without obtaining any useful results.
In fact, at this moment,the scientific community is putting a lot of effort into finding a fast and affordable sensoristic system , and yet no definitive solution has been reached.

Discovering this interaction between the GV38 molecule and some PFAS can serve as a foundation for many future studies, both with new molecules and new techniques to further improve the data. For instance, we have already seen promising results when performing Electrochemical Impedance Spectroscopy (EIS) with a Redox couple, which requires further analysis, and many more tests will be necessary to consolidate and characterize the results obtained so far.

DBTL cycles

Our work, being a synthetic biology project, has seen the engineering approach being applied in every step we took, from designing biological parts to planning new strategies.
Documenting our trials and errors and what we have learned from them is not only useful to us, but also to future teams that will participate in iGEM’s competition in the following years. This helps them to prioritize the use of certain technologies or techniques that can be favored despite others , or some mistakes can be avoided from the start.
The fact that DeHa2 was considered probably toxic to our chosen host E. coli not only made us consider the idea of switching the strain in which we tried to clone our vectors, but also made us think about what other measures needed to be taken to express our protein without causing excessive cellular death: that’s why we tested transformed E. coli’s growth when protein expression is induced at different IPTG concentrations.
Thanks to the enzymatic tests we performed, we were also able to see that Lpp-OmpT-DeHa2 can potentially degrade chloroacetate, its natural substrate and we verified dehalogenase activity; even if these data are still preliminary and need in-depth further evaluation, they pave the way for promising evolutions in future experiments.

The approach that characterizes the field of synthetic biology (Design - Build - Test - Learn) underpins all SurPFAS, including sensor and filter modules.
In the latter part of surPFAS we developed two main strategies: seeking an adsorbent with greater capacity than GAC to reduce replacement frequency and environmental impact, and exploring a chemical regeneration technique as an alternative to thermal methods. Documenting our trials and insights is essential not only for our understanding but also for future teams tackling similar challenges. This documentation will help others prioritize effective technologies and avoid common pitfalls.

During our testing, we evaluated various desorption solutions for GAC and found that the ones tested for GAC were not suitable for industrial application (because of the need for elevated volume of methanol or ethanol); we then investigated Anionic Exchange Resins (AERs) which demonstrated high adsorption capacity for PFAS, so they present themselves as possible alternatives to GAC, moreover these adsorbent can be regenerated with a solution of NaCl so it is not harmful, but the results obtained in the laboratory are not sufficient to guarantee good efficiency of the regeneration process.
This insight underscores the importance of further research into alternative desorption solutions to optimize our approach. Our findings lay the groundwork for advancements in future studies focused on PFAS remediation.
From the DBTL cycles we performed during the development of our sensor, we believe the most interesting takeaway is the possibility of using the same surface for different analytical techniques, allowing us to combine their advantages.
In our case, by developing a surface capable of attracting various types of PFAS, we would be able to use the EIS technique to quickly perform simple field analyses, providing a result that indicates a 'general quantity' of PFAS adhered to the surface (although SERS can also be applied in the field, EIS remains simpler and faster)and with the SERS technique, we would be able to differentiate between the various compounds that have adhered.

Further information regarding our engineering success can be found in Engineering and in Results pages.

Growth tests

Developing our project, we decided to characterize the survival of E. coli in the presence of substrates that could end up in the bioreactor, to asses the fitness of this chassis for working in the desired scenarios.
Firstly, we needed these tests to understand the best achievable condition inside the bioreactor that would not only contain PFAS molecules, but also the desorption solution itself.

Many of the collected results can be useful to future teams researchers, so here we reported an overall review of what we achieved.

In the following table we present at what concentration of each component that characterized the test, E. coli’s growth was inhibited by about 60% when compared with the negative control.

All these tests were done at the plate reader measuring the OD at 600 nm.

Methanol: from 0 to 2.75 M methanol causes a decrease in E. coli’s growth but does not inhibit it completely.

Ethanol: it has a stronger antimicrobial effect when compared with ethanol. There is a decreasing trend on the growth as the concentration increases and from 1M, the bacterial growth is inhibited by the 60%.

NaCl: it causes a drastic decrease in bacterial growth for concentrations higher than 2% kg/L.

PFOA: Cultural mediums containing PFOA cause a decrease in E. coli’s growth and it is almost completely inhibited by 15 mM in every condition that has been tested: PFOA with LB, PFOA with methanol and PFOA with ethanol. PFOA with NaCl 2% needs further investigations.

PFBA: Cultural mediums containing PFBA cause a decrease in E. coli’s growth. Differently to PFOA where the growth is inhibited for concentrations over 10 mM, the bacterial growth in the presence of PFBA seems sometimes to withstand up to 15 mM included.
For both PFOA and PFBA, usually between 10 mM and 15 mM is the frame where there is the most sudden decrease in E. coli’s growth.

Chloroacetic acid: We tested concentrations of chloroacetic acid between 0 and 3.5 mM. The results of our tests show no apparent difference in E. coli’s survival at those concentrations.

A more in-depth presentation of our results can be found on the Results page.

Business plan

Incorporating an entrepreneurial perspective into the project was crucial for advancing research towards practical applications. Developing a complete and detailed business plan became essential, shifting the project from a purely research-based initiative to a viable, real-world solution. We are proud to present our market analysis to the scientific scenario, hoping to inspire other bioremediation projects to come to light outside of the laboratory. Check out our entrepreneurship page to learn more about our business plan.

Community Engagement

When one of our team members who lives in Veneto’s red area was called to participate in the health screening indicated by the Veneto Region itself, she found out that she had an extremely high PFAS concentration in her blood. By informing ourselves and the remediation methods in place were not leading to an actual degradation of the compounds, our team knew we had to put our minds together and focus on the PFAS problem.

We contacted professors, companies (such as water service providers in our region) to better understand the technologies currently used to treat these pollutants. We also met with an activists’ group and the inhabitants of the Red Zone, helping us redirect our project according to their needs.

These activities were crucial in understanding the best way for a scientist to have successful communication with society, and we are enthusiastic to share our experience with the whole scientific community. Check out our Integrated Human Practices page.