iGEM 2024 — Team uni-padua-it

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block page\_content %}

# {{ self.title() }}

{% block lead %}{% endblock %}

| Name | Link |
| --- | --- |
| Cloning lab 1: protocols | [Link 1](https://static.igem.wiki/teams/5109/notebook/cloning-lab-1-protocols-docx.pdf) |
| Cloning lab 1: notebook | [Link 2](https://static.igem.wiki/teams/5109/notebook/cloning-lab-1-notebook.pdf) |
| Cloning lab 2: protocols | [Link 3](https://static.igem.wiki/teams/5109/notebook/cloning-lab-2-protocols.pdf) |
| Cloning lab 2: notebook | [Link 4](https://static.igem.wiki/teams/5109/notebook/cloning-lab-2-notebook.pdf) |
| Growth tests lab: notebook | [Link 5](https://static.igem.wiki/teams/5109/notebook/notebook-enzymatic-tests.pdf) |
| Enzymatic tests: notebook | [Link 6](https://static.igem.wiki/teams/5109/notebook/notebook-growth-tests.pdf) |

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block page\_content %}

# {{ self.title() }}

{% block lead %}{% endblock %}

Synthetic biology is a relatively recent and fast-developing interdisciplinary
field that combines different branches of biological sciences with an engineering
approach, with the main goal of designing new biological parts, devices,systems
or redesigning existing ones. [1]
Engineering contributes to this rapidly growing field by introducing the concept of
modularity and promoting a systematic approach to both the creation of new biological
components and the design of experiments and processes: the engineering cycle.
This approach consists of four different stages, design, build, test, and learn which are supposed to be repeatedly iterated until the developed systems are sufficiently matching the desired project specifications.

![](https://static.igem.wiki/teams/5109/engineering/engineering1.png)

In our project, this cycle was applied to every step, from designing our intracellular and extracellular expression cassette to setting up new protocols and new experiments both in wet and dry labs.

* Wet lab
* Sensor
* Filtration

* [Clonings](#Titolo1)
  + [Cycle A](#Titolo1-1)
  + [Cycle B](#Titolo1-2)
  + [Cycle C-1](#Titolo1-3)
  + [Cycle C-2](#Titolo1-4)
* [Protein expression analysis](#Titolo2)
* [Enzymatic tests](#Titolo3)

# Clonings

## Cycle A: introduction of gel-extraction in our protocols

**Design**:
 We tried to build our expression vector by inserting our new composite part ([BBa\_K5109001](https://parts.igem.org/Part%3ABBa_K5109001)) in pJUMP29-1A plasmid backbone using the BioBrick standard.
We opted for digesting both insert and vector with EcoRI and PstI, and soon after we proceeded to clone our new expression vector inside the E. coli DH5α strain.
before starting with cloning experiments, we designed our cloning protocols in the following way:

* digestion with EcoRI and PstI of both vector and insert
* PCR clean-up of the insert digestion result
* dephosphorylation of pJUMP29-1A digestion to avoid self-ligation
* ligation
* cell transformation in E. coli DH5α
* plating on petri dishes with LB and kanamycin
* colony picking and overnight growth in liquid medium
* colony PCR with primers mapping on the prefix and suffix regions
* sequencing of plasmids coming from colonies that gave a positive outcome

**Build**:
 we applied the previously designed protocol to perform our experiments.

**Test**:
 transformed cells were plated on kanamycin agar plates to select bacteria
The colony PCR result of the few colonies that were able to grow on our kanamycin plates was then run on an electrophoresis gel. The gel always showed the presence of a 500bp band, which meant that the plasmid contained the original insert between the prefix and suffix regions.

**Learn**:
 all vectors with the original insert were partially digested plasmids that self-ligated, indicating that the plasmid dephosphorylation step was not efficient enough.To tackle this problem, we opted for switching to a different technique to isolate our digested vector: Gel extraction. We performed gel electrophoresis on the pJUMP29-1A digestion product, isolated the plasmid band at 2900bp, and extracted it from agarose with gel-ext kits. This allowed us to effectively separate the original insert from the digested plasmid.

## Cycle B: transformation in a E. coli strain with the constitutive expression of LacI

**Design**:
 In the beginning, we planned to use the pTac as a constitutive promoter and then add a constitutive LacI expression cassette to make our protein of interest’s expression inducible with IPTG.

After many failed attempts, we hypothesised that DeHa2 may be toxic for E. coli. Some successful ligation products may have entered our chemically competent cells, but those cells may have not survived due to the fact that the expression of our heterologous protein was too high for a constitutive pTac promoter not blocked by Lac repressor.

Instead of trying to insert the LacI expression cassette in our genetic construct, making it more complex and harder to clone than it already was, or co-transform our expression vector with an another backbone containing the LacI gene, risking to increase cells’ metabolic burden, we decided to switch to an E. coli strain containing an episome that allowed the constitutive expression of LacI, Top10 F’.

**Build**:
We digested pJUMP29-1A and our intracellular expression cassette containing starting enzyme DeHa2 with the aforementioned restriction enzymes EcoRI and PstI, we performed electrophoresis and gel extraction on the digestion result of both the plasmid and the synthetic insert to remove the original plasmid insert between biobrick prefix and to maintain both digestion products sticky ends, and we then transformed chemically competent Top10 F’ cells.

**Test**:
The transformation result was plated on a LB petri dish with kanamycin and we saw some colonies the next day, so we picked all colonies grown and we ran a colony PCR.

**Learn**:
All the picked and tested colonies still gave us a negative result, meaning that changing host was not sufficient to achieve cloning success.

However, we decided to stick with this idea as avoiding cloning an additional LacI expression cassette would simplify our cloning strategy and avoid burdening our cells with plasmidic co-transformation.

This choice was revealed to be the correct decision later on, when we changed our set of primers as described in cycle C-2: this step was crucial for improving our experimental design’s efficiency by shortening the amount of trials to reach a positive result.

## Cycle C-1: a matter of primers

**Design**:
Two different primers mapping on Biobrick prefix and suffix were designed to amplify our synthetic intracellular expression cassette, so that we could use it for clonings later on.

Primers’ sequences are:
Prefix\_fw: 5’- GAATTCGCGGCCGCTTCTAGAG -3’
Suffix\_rv: 5’- CCTGCAGCGGCCGCTACTAG -3’

**Build**:
The synthetic sequence was amplified with PCR, then digested and used throughout different cloning attempts.

**Test**:
Transformation results were plated on a LB-agar Petri dish with kanamycin. The colony PCR result of the few colonies that were able to grow on our kanamycin plates was then ran on an electrophoresis gel. The gel always showed the presence of a 500bp band, which meant that the plasmid contained the original insert between the prefix and suffix regions.

**Learn**:
After many failed attempts, we hypothesised that maybe the ligation did not occur due to either the vector or the sequence not being correctly digested by EcoRI and PstI. Therefore we analysed the primers that we used to amplify the sequence, and decided to create new ones with a bigger amount of landing bases that could increase the probability of digestion to happen.

## Cycle C-2: a matter of primers

**Design**:
Given the negative outcome of the previous attempts, as mentioned above we designed a new set of primers to amplify our synthetic sequences that had the following sequences

fw: 5’- CATCATGGAATTCGCGGCCGCTTC -3’
rev: 5’- CAAGCCCTGCAGCGGCCGCTA -3’

![](https://static.igem.wiki/teams/5109/engineering/engineering2.png)

![](https://static.igem.wiki/teams/5109/engineering/engineering3.png)

Due to time limitations, we decided to amplify with this new set of primers and perform cloning attempts only with our extracellular expression cassettes.

**Build**:
Our synthetic sequences were amplified by performing PCR using a High Fidelity Taq polymerase and then subsequently digested with EcoRI and PstI and cloned into pJUMP29-1A.

**Test**:
Transformation results were plated on a petri dish with kanamycin to see whether there were any transformed cells. As before, we picked the colonies grown on the plate and set up a colony PCR and sent them to the sequencing facility.

**Learn**:
After a few attempts, we were able to see colonies that tested positive for the insertion of our Lpp-OmpT-DeHa2 expression cassette ([BBa\_K5109023](https://parts.igem.org/Part%3ABBa_K5109023%3AExperience)) and Lpp-OmpT-Dehalogenase S ([BBa\_K5109020](https://parts.igem.org/Part%3ABBa_K5109020)) expression cassette,
therefore confirming our cloning success as seen in our [results](https://2024.igem.wiki/Uni-Padua-IT/results) page.

Three hypothesis we elaborated to explain our cloning success and they are the following:

* Our clonings were previously failing due to landing sequences being too short for the activity of restriction enzymes, leading to no digestion activity
* The new host cells had a constitutive repressor that may have helped mitigate the toxicity of over-expressed enzymes.
* Surface expression of our enzymes may be less toxic than the intracellular one, therefore causing less bacteria to die after transformation has occurred.

To verify this last point, whole-cell SDS-PAGE and subsequent Western Blot assays should be performed to quantify protein expression.

To assure its correct exposure on the cell surface, we can subject our engineered bacteria to a thrombin treatment. Thrombin is a proteolytic enzyme that cleaves at its recognition aminoacidic sequence. In our design, it is collocated between the anchoring motif and the exposed protein allowing release of our enzymes in the culture medium.

Further SDS-PAGE and Western blot assays on the medium may help us understand if we have surface displayed proteins that were cleaved.

# Protein expression analysis

### CYCLE A: IPTG concentration to induce protein expression

**Design**:
 Since having a positive cloning result with Lpp-OmpT-DeHa2, a growth test with increasing concentrations of IPTG was needed to perform enzymatic tests and future SDS-PAGE assays. Since the pTac promoter that allows the expression of the dehalogenase is inducted by the presence of IPTG and, by this time, we were not sure about the toxicity of the dehalogenase, we wanted to understand what is the best IPTG concentration induction that less damages bacterial growth and allows a sufficient expression of the protein.

**Build**:
 liquid cultures of colonies #4 and #9 retrieved from the Lpp-OmpT-DeHa2 successful cloning and wild type Top10F’ were plated on a 96 multiwell plate.

For this test were prepared culture media containing increasing concentrations of IPTG: 0,5 μM, 5μM, 50 μM, 500μM.

**Test**:
cellular growth was measured using a plate reader by taking the OD600 value every five minutes for 14 hours.
We then used the matrix of data generated by the plate reader to create the growth curves and analyze how they changed in the different concentrations of IPTG.
The curves obtained from the analysis are the following:

![](https://static.igem.wiki/teams/5109/engineering/engineering4.png)

![](https://static.igem.wiki/teams/5109/engineering/engineering5.png)

The colony number #3 showed a significantly different growth in the 500 uM medium, therefore we decided to treat it separately in the creation of the curves and in the data analysis

![](https://static.igem.wiki/teams/5109/engineering/engineering6.png)

**Learn**:
We noticed a significant difference between the growth of the two colonies transformed with the Lpp-OmpT-DeHa2 construct.

Although both colonies grew slower than the wild-type, we saw that the growth of colony #4 was strongly inhibited for concentration of IPTG higher than 5 μM, while the growth of colony #9 was only slightly affected by the variation of IPTG concentrations.

From this data we deduced that the standard IPTG concentration of 500 μM was not suitable for our protein of interest’s induction in the colony #4, since it led to dosage of protein production that was lethal for our host, but could be used for the colony #9.

The sequencing that we obtained for these colonies had a really poor quality, therefore we could not make any assumptions about the genetic differences between the colonies, and we were not able to justify the differences in the growths.

For this reason and for the shortage of time, we decided to make a conservative choice and use for our enzymatic tests the lowest concentration that could be suitable for both the colonies, being the best compromise between having the biggest hypothetical amount of expressed Lpp-OmpT-DeHa2 and having the smallest effect on E. coli’s growth, which corresponded to 5 μM.

Further investigation with more colony sequencing and protein expression analysis with experiments such as SDS-PAGE and western blot should be performed to properly understand the differences between the colonies.

# Enzymatic tests

### Cycle A: Enzymatic tests for dehalogenase

**Design**:
The first step for the expression of a desired protein is to design the construct that will be inserted in an expression plasmid and transformed into bacterial cells. The main goal of our project is to enable surface expression of a dehalogenase in our chosen host E. coli. This can be done by fusing the DeHa2 sequence at the C-terminal of an anchoring motif, in our scenario the Lpp-OmpT surface display system, that contains a signal peptide that allows correct transport and collocation of the fusion protein and a transmembrane domain.

Overall, our construct leads to the expression of a chimeric protein made up of a carrier protein, the anchoring motif, and a passenger enzyme, DeHa2, controlled by the inducible promoter pTac.

**Build**:
After designing and synthesising the construct, it was ligated inside a pJUMP plasmid that had been previously digested. After ligation, the engineered plasmid was transformed inside Escherichia coli Top 10F’ and at the end of the process it was verified that the colonies that were obtained had the desired construct.

**Test**:
After the cloning part, our next step was to verify the correct activity of the enzyme that we decided to express: DeHa2. DeHa2 is a dehalogenase, an enzyme that is able to remove halogens bound to a C atom.

The enzymatic activity was measured by introducing chloroacetic acid 2.5 mM in our medium and measuring its degradation by taking a sample at three timepoints (0, 24 and 48 hours) and measuring via FIA-HRMS (flow injection analysis - high resolution mass spectrometry).

**Learn**:
The results showed no significant differences in the chloroacetic acid concentration at the three timepoints in the Top10 F’ wild type and in the colony #4 transformed with the Lpp-OmpT-DeHa2 construct. In colony #9 a total degradation of the reagent was measured instead, even when the protein expression was not induced.

Details on why there is a marked difference between the two analysed colonies needs further research but from this test we still achieved many new understandings.
Firstly, the fact that colony #9 was able to almost completely degrade chloroacetic acid even when not induced, may be an indicator of the leakage of the pTac promoter. This means that, even without the inductor, the protein gets expressed and is able to remove halogens.
Secondly, there needs to be further research on each of the constructs of those colonies to understand if there are any differences in the gene sequences or in the reading frames and to make further assumptions.

Further turns of the engineering cycle are being performed to address these issues, unfortunately not in time for the wiki freeze.

* [Cycle A](#TitoloA1)
* [Cycle B-1](#TitoloA2)
* [Cycle B-2](#TitoloA3)

A more in-depth analysis of the sensor construction processes is reported in
[this section](https://2024.igem.wiki/Uni-Padua-IT/sensor).

# Cycle A

**Design**:
The first prototype we used to attempt our measurements consisted of a simple gold nanoparticles (Au-NPs) surface, designed to allow us to use both measurement techniques.

**Build**:
After a 5-hour liquid ablation process, we obtained a solution containing the right concentration of Au-NPs. Then, we started a spray coating process using an airbrush on a glass plate. To create a more defined surface to work on, we used a mask over the plate during the process.

**Test**:
We performed measurements on this first prototype using the Surface Enhanced Raman Scattering (SERS) technology, both by solution analysis with a containment chamber and via drop-casting. We analysed PFOA and PFOS.

**Learn**:
The solution analysis did not produce any interesting signal via SERS. With drop-casting, we confirmed that there was no adhesion of PFAS molecules on the surface, as they seemed to prefer aggregating among themselves and crystallising during the evaporation of the drop. When pointing the laser for SERS analysis on these crystals visible under the microscope, we obtained good Raman signals. Pointing it towards other parts of the surface showed no new Raman peaks. We concluded that it is necessary to add a functionalization molecule to the surface to allow PFAS adhesion.

# Cycle B-1

**Design**:
Based on the results of cycle A, we considered molecules that might work for our purpose. We identified 11-mercapto-1-undecanol as a possible candidate. It has a Tiole group (-SH) capable of bonding well to the gold nanoparticles present on our surface, an elongated carbon chain structure and a positively polarised functional group that could interact with PFOA molecules. The PFOA molecule has a slightly negative polarity.

**Build**:
After carrying out the usual Au-NPs surface development process as described in cycle A, we functionalized the surface. Functionalization was performed by incubating the surface in a solution of methanol and our molecule for 1 hour to ensure proper adhesion.

**Test**:
We performed Electrochemical Impedance Spectroscopy (EIS) and SERS analysis on the surface to verify the correct adhesion of the functionalization molecule and then tested with a solution containing 1mM PFOA in milliQ water at various time intervals. To perform the EIS analyses, 10mM NaCl was added as the electrolyte.
SERS results showing a small variation from the bare, probably due to a small PFOA-surface interaction. 785 nm wavelength, 10X, 0.1 % power, 6x30s acquisition. The graphic is the following:

![](https://static.igem.wiki/teams/5109/engineering/engineering7.png)

**Learn**:
We obtained excellent signals from the functionalization molecules with both techniques, confirming their correct and stable adhesion to the surface. However, we observe a very small variation indicating PFOA adhesion is happening, but needs further improvement. We realised that while the Tiole group allowed very stable adhesion of the molecules to the surface, further assistance was needed on the other end of the chain to promote the approach of PFOA molecules.

# Cycle B-2

**Design**:
The results of test B-1 showed us that PFOA molecules do not interact with the detection surface. Thanks to collaboration with a student from our university, we obtained a new functionalization molecule (referred as GV38) that has a shape more similar to PFOA. It still features a Tiole group, which we had found effective for adhesion to the gold surface in cycle B-1. This time the molecule has a C9 carbon chain and a Positive Guanidinium group for interaction with PFOA. We also changed the electrolyte in the test solutions, replacing NaCl. We chose Hepes because this compound acts as a buffer and keeps the pH stable at 7.55, a value we believe can facilitate the proper interaction of PFOA with the surface.

**Build**:
As described in the previous cycles, we performed the gold surface creation process. This time, the functionalization solution was modified and now consists of MeOH and GV38 1mM.

**Test**:
We conducted our tests using EIS and SERS techniques on the surface before and after functionalization, and then after adding the solution with milliQ water + PFOA 1mM + Hepes 10mM. SERS test is made with 633 nm laser wavelength. It detects PFOA molecules thanks to the presence of two characteristic C-F bonds peaks in the Raman spectra. The graphic is the following:

![](https://static.igem.wiki/teams/5109/engineering/engineering8.png)

EIS results showing an impedance variation due to the attachment of PFOA molecules to the detection surface. The graphic is the following:

![](https://static.igem.wiki/teams/5109/engineering/engineering9.jpg)

**Learn**:
The results were optimal with both techniques, confirming a stable long-term interaction between our functionalized surface and the PFOA molecules. From this point we can proceed to further characterise this result and try it under different conditions.

* [Cycle A](#TitoloB1)
* [Cycle B](#TitoloB2)

In order to cope with PFAS pollution, water service providers utilize filters composed of granular activated carbon (GAC) which adsorbs PFAS molecules on its surface.
When GAC reaches its PFAS sorption capacity it must be removed and replaced with a new one or the treatment system will not remain effective. Exhausted GACs are replaced every two or three months with virgin ones because the only
available reactivation technique is thermal, with temperatures up to 900°C, which lowers GAC sorption capacity.

For a more in-depth description visit
[Plant Design - Filters](https://2024.igem.wiki/Uni-Padua-IT/filters).

# Cycle A: Investigation of GAC chemical regeneration

**Design**:
To change the current situation we wanted to find a regeneration technique alternative to the thermal one; we opted for a chemical regeneration through the use of a desorption solution. After a review of the literature we decided to analyse two possible desorption solutions for GAC, one with methanol and NaOH and one with ethanol.

**Build**:
The desorption solutions analyzed were: MeOH : H2O + NaOH 0.1 M, 50:50 and EtOH : H2O, 50:50 with 2 possible GAC mass - desorption volume ratio (200 mg of GAC with 20 mL of solution and 10 mg of GAC with 100 mL of solution).

**Test**:
To analyze solvent regeneration of GAC we made 6 tests to compare the effectiveness of wet adsorbent with a dried one, select one of two possible GAC mass - desorption volume ratio and decide on the best desorption solution between the two proposed.

![](https://static.igem.wiki/teams/5109/engineering/engineering9.png)

**Learn**:
Through the data obtained from our GAC desorption experiments we understood that the solutions proposed have not the efficacy required to be applied to an industrial context; moreover a scale-up of this chemical desorption technique would require elevated volume of methanol or ethanol, posing an environmental and economical issue (for the economical aspect visit
[Entrepreneurship](https://2024.igem.wiki/Uni-Padua-IT/entrepreneurship)
). Further studies should be conducted on the subject, analyzing other organic and non-organic solutions and focusing on their effect on Filtrasorb 400, the GAC type used commercially. In addition, it is important to bear in mind the need to have low volumes of possible toxic solutions used in the scaleup system so as to be environmentally friendly and economically feasible.

# Cycle B: Investigation of AER as an alternative to GAC

**Design**:
We then focused our attention on finding an adsorbent with higher sorption capacity than GAC and that can be regenerated using a non-organic solution. This would reduce the need to replace filters so frequently, reducing costs and environmental impact.

**Build**:
We focused on AER (anion exchange resins) as multiple studies find them to have high sorption capacity and we found out it could be regenerated using a desorption solution made with a 10% w/V NaCl solution.

**Test**:
 To understand the adsorption capacity of the resins selected (A860, A110, A111) we put them in contact with PFOA and PFBA solutions (CPFOA=3,75 \* 10-3 mol/L and CPFBA=5,27\* 10-3 mol/L ) for 2 hours and measured the concentration of pollutants every half hour.

![](https://static.igem.wiki/teams/5109/engineering/engineering10.png)

To analyse the intended AER chemical regeneration, we adopted the NaCl solution for the 3 investigated resins.

![](https://static.igem.wiki/teams/5109/engineering/engineering11.png)

**Learn**:

* The AER adsorption experiments lead us to demonstrate the extremely high PFAS-adsorption properties of these materials, such that it is difficult to reach an equilibrium state and thus effectively determine their absorption capacity; further studies on this topic are needed.
* The AER desorption tests did not confirm the data collected in the literature. We assume it is due to the difficulty in reproducing their experimental conditions. As a result, it is critical to perform further experiments to prove those data.

## Let's find out more about our project

### Project

[![Description Icon](https://static.igem.wiki/teams/5109/footer/description-icon.svg)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)
[Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)

[![Design Icon](https://static.igem.wiki/teams/5109/footer/design-icon.svg)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)
[Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)

### Wet Lab

[![Lab steps Icon](https://static.igem.wiki/teams/5109/footer/lab-steps-icon.svg)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)
[Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)

[![Results Icon](https://static.igem.wiki/teams/5109/footer/results-icon.svg)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)
[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block page\_content %}

# {{ self.title() }}

{% block lead %}{% endblock %}

# Summary

During this year, we aimed to express four chosen enzymes into an E. coli strain, since it is the most practical and known bacteria, and test their expression and functionality in this chassis: this was a necessary step since three out of our four chosen enzymes are not native to E. coli species and the effects of this change of host are not yet known.
To achieve this, we designed and synthesized novel expression cassettes codifying specific dehalogenases or laccases that will be expressed internally or externally, through the insertion of the display system Lpp-OmpT, on the E. coli strain. After, expression and functionality tests were performed to verify the correct expression of the translated protein, ideally
comparing enzyme localization both inside and outside the bacteria membrane, and the enzymatic activity of each protein through the use of a known substrate.

# Premises

1. DNA extraction and purification from bacterial colonies were performed via the QIAprep Spin Miniprep Kit.
2. Plasmid gel extraction upon electrophoresis was performed using the QIAquick PCR & Gel Cleanup kit.
3. The concentration of DNA samples was quantified via Thermo Fisher NanoDrop.
4. All synthetic sequences (expression cassettes and primers) were synthesized by Integrated DNA Technologies (IDT).
5. After every cloning attempt, the product was sent to BMR Genomics for Sanger sequencing.
6. Every synthetic sequence was designed using the Benchling platform.

## pJUMP29-1A NdeI site MUTAGENESIS

The chosen backbone pJUMP29-1A was fundamental for our clonings since it presents a terminator right after the Biobrick suffix. Since the synthesis facility wasn’t able to synthesize an expression cassette that included a terminator itself, the presence of this Lambda terminator downstream of the Suffix element came in handy for designing and building our system.

During the design of our basic and composite parts, we decided to include in our sequences the NdeI restriction site, formed by a six-base sequence “CATATG”. This particular site was chosen for different possible procedures, but it is always necessary since the “ATG” triplet in the recognition site overlaps the starting codon ATG of our coding sequences, and is needed due to its closer position to the start of the gene.
At this point we faced a problem: pJUMP29-1A presents an NdeI site inside its sequence.
So our solution was to mutate the backbone through mutagenic PCR to remove the unwanted site.

The restriction site mapped on the backbone but was outside of Ori and the kanamycin resistance gene, so it was easier to modify without damaging the plasmid structure.
The sequence that encodes the site was:
5’–CCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAG–3’

We designed two primers forward and reverse overlapping the restriction site, both of them with a single mismatch on the second T of the sequence:

* Primer forward: 5’ – CCGCATAAGGTGCACTCTCAGTACAAT – 3’
* Primer reverse: 5’ – GTGCACCTTATGCGGTGTGAAATACCG – 3’

With these, we performed a mutagenic PCR.

At the end of the procedure, the amplicon was purified with a PCR purification kit and it was digested with the enzyme DpnI, to remove every original plasmid sequence. DpnI is a restriction enzyme that cleaves methylated DNA only: DNA methylation is a process in which methyl groups are added to a DNA sequence, and it’s a process that can happen exclusively when DNA is replicated inside a living host.
By eliminating methylated DNA sequences, we eliminate the majority of parental copies of pJUMP29-1A, eliminating all the non-mutagenized sequences. The DpnI-treated PCR product is incubated with PNK later on to improve ligase activity. Ligation was carried on overnight.

To see whether mutagenic PCR was effective, a part of the amplicon was digested with NdeI and run in gel: the gel run showed promising results when comparing the digested plasmid, which was linearized, to the control plasmid left uncut.

Finally, a part of the amplicon was digested with NdeI and run in gel to verify the success of the mutagenesis.

## CLONING OF INTRACELLULAR DEHA2

The starting point of our project was to insert the expression cassette for the intracellular enzyme DeHa2 (Part
[BBa\_K5109001](https://parts.igem.org/Part%3ABBa_K5109001)) into our chosen E. coli strain, DH5α, as it was already widely used in our laboratory and it’s very efficient in the expression of orthologous proteins.

![](https://static.igem.wiki/teams/5109/experiments/experiments1.png)

After the sequence arrived at our lab, we started our cloning protocols: to insert our intracellular expression cassette containing DeHa2 (Part
[BBa\_K5109001](https://parts.igem.org/Part%3ABBa_K5109001)) into the pJUMP29-1A vector (Part
[BBa\_J428341](https://parts.igem.org/Part%3ABBa_J428341)), our designed backbone, we used the digestion-ligation method with the BioBrick prefix and suffix.

1. The first thing we did was to digest, at the same time, the expression cassette and the pJUMP29-1A vector with the restriction enzymes EcoRI and PstI, both at the further ends of the BioBrick prefix and suffix respectively.
   This way, the construct is inserted in a directed manner and the BioBrick prefix and suffix will be restored.
   We proceeded to carry out the reaction at 37°C for 1:30 h, then we inactivated the enzymes at 80°C for 20 minutes. When both reactions were inactivated, we ran a gel electrophoresis to verify the correct digestion of our backbone: it was not possible to check the digestion outcome for the insert, as the terminal ends resulting in successful digestion were only 20 bp long and would not be visible on the gel.
2. When the digestion was confirmed, we purified the digested [BBa\_K5109001](https://parts.igem.org/Part%3ABBa_K5109001) using the Qiagen purification kit and quantified the result, meanwhile, we dephosphorylated the backbone using an alkaline phosphatase, as to prevent the re-closing of pJUMP29-1A without any insert. The reaction was carried out at 37°C for 20 minutes and the phosphatase was inactivated at 80°C afterwards.
3. Then, we set up a ligation protocol: we decided to use a 5:1 ratio, meaning that the concentration of the insert in this reaction was five times higher than the concentration of the backbone; this was done to maximize our possibilities of a successful ligation.
   We prepared two different ligation reactions, one of them containing only the dephosphorylated backbone without the insert: this reaction was created to act as a control, as after the transformation of our two ligation reactions in E.coli there should be a clear difference in numbers of colonies grown from one plate to the other.
4. We left our ligation reaction at 16°C overnight, then we transformed them into chemically-competent DH5α cells: this was done via heat shock, where pores are formed on the bacterial membrane after a sharp change in temperature (from 42°C to 0°C) so that the external DNA can enter the bacteria. When the heat shock was complete, we left the bacteria to recover in LB at 37°C for an hour, then we plated them in LB agar with kanamycin, the selection marker present in our backbone
   [BBa\_J428341](https://parts.igem.org/Part%3ABBa_J428341).
   When the colonies were visible, a clear distinction in number was seen between the two plates: the control plate showed a lot less of colonies than the ligation plate, making us think that the lack of insert didn’t help the growth of viable colonies and that the ligation plate contained complete and recombinant plasmids.
5. We picked a handful of colonies from both plates and we run a colony PCR, using a set of primers designed to bind on the BioBrick Prefix (forward primer) and Suffix (reverse primer): this way, It's possible to visualize the result of the PCR amplification on gel, where a band 800-900 bp long should be visible if our desired expression cassette has been correctly cloned.

From now on, to have a better chance to perform clonings successfully, we changed the E. coli strain used. We switched to a different strain, called Top10 F’: this strain can constitutively express the LacI repressor present in the F’ episome, an important component to the regulation of the Lac operon. With this change, our aim was to repress any expression of our protein of interest simplifying clonings and experiments.

## HOMOLOGOUS RECOMBINATION

During the first two months of work, we couldn’t get any results from the cloning trials. So we decided to give a chance to a different technique that uses homologous recombination. In this technique, both vector and insert are amplified by PCR with primers that have overlapping ends, and E. coli’s ability to perform in vivo homologous recombination allows for our insert and vector’s extremities to pair, be cut and then fused together, obtaining a closed vector. [1]

![](https://static.igem.wiki/teams/5109/experiments/experiments2.png)

The purpose of the work was to clone the DeHa2 type 2 intracellular expression tool (Part
[BBa K5109001](https://parts.igem.org/Part%3ABBa_K5109001)), inside our chosen backbone pJUMP29-1A. The first strategy of RFC 10 cloning wasn’t showing any results, so we designed and ordered specific primers to perform the recombination.

1. The protocol includes a PCR amplification as the first step, to amplify the desired insert and vector with primers including identical ends to both of them. After the end of the PCR and the purification of the product, both of the amplicons will present the same ends and will be in a linear form.
2. Vector and insert will be mixed together in a solution of 3:1 insert to vector proportion, and this preparation will be used to chemically transform competent cells.
3. After transformation, if recombination occurs, cells are plated and grown overnight on LB + Agar + antibiotic (kanamycin, in our case). The day after, colonies will grow only if homologous recombination has occurred, since they will be equipped with a plasmid carrying antibiotic resistance.
4. We relied on Top10 F’ strains for this cloning, even if they present a deletion of RecA in order to avoid nonspecific recombination, since this type of homologous recombination is independent from the presence of RecA but it instead exploits different cellular mechanisms.

## CLONING of EXTRACELLULAR DEHA2, EXTRACELLULAR DEHALOGENASE S and EXTRACELLULAR LACCASE S

After our first unsuccessful attempt, we tried to clone three new expression cassettes, this time for the extracellular expression of DeHa2 ([BBa\_K5109023](https://parts.igem.org/Part%3ABBa_K5109023)), Dehalogenase S ([BBa\_K5109020](https://parts.igem.org/Part%3ABBa_K5109020)) and Laccase S ([BBa\_K5109022](https://parts.igem.org/Part%3ABBa_K5109022)): all of these cassettes contain a Lpp-OmpT-linker part ([BBa\_K5109004](https://parts.igem.org/Part%3ABBa_K5109004)) to create a display system for the enzymes, placed upstream of the GOIs. We cloned them simultaneously, using the same protocols and reagents, so that all three of them had the same chance of succeeding.

![](https://static.igem.wiki/teams/5109/experiments/experiments3.png)

![](https://static.igem.wiki/teams/5109/experiments/experiments4.png)

![](https://static.igem.wiki/teams/5109/experiments/experiments5.png)

Since time was starting to become a limiting factor, we decided not to try the intracellular expression of the enzyme DehalogenaseS (BBa\_K5109013) and Laccase S (BBa\_K5109015) and to instead try directly the extracellular expression using the display system Lpp-OmpT, so that it would be a more interesting study during our expression and functionality assays.

1. We digested BBa\_K5109023 (DeHa2 extracellular expression cassette), BBa\_K5109020 (Dehalogenase S extracellular expression cassette), BBa\_K5109022 (Laccase S extracellular expression cassette) and BBa\_J428341 (pJUMP29-1A) with the restriction enzymes EcoRI and PstI, so that the three cassettes would have matching ends with the digested pJUMP and would pair up without problems.
   The digestion of the synthetic expression cassettes was performed as illustrated during the cloning of BBa\_K5109001, but something different was done for the digestion of the backbone: the digestion was done in a final volume of 60 ul, then, after the enzymes were inactivated after 2h at 37°C, all the digested backbone was run in gel electrophoresis. The gel was also made in a different way: we usually set up the gel at 1% agarose concentration and using Midori Green as our intercalating agent, but we prepared this gel at 0.7% agarose concentration, to better separate the different bands of the digestion result and the ladder. We also used ethidium bromide as our intercalant agent, as to better visualize the different bands on the gel.
2. After the digestion was completed and the gel electrophoresis had stopped running, we performed a gel extraction to gather our digested backbone: this was done to ensure that no original insert was present in the subsequent steps and to minimize the chance of another negative result. The gel extraction was performed using the QIAquick Gel Cleanup kit and we quantified the result afterwards; meanwhile, we purified our digested expression cassettes using the Qiagen purification kit, and we later quantified the obtained results.
3. When both the backbone and inserts were ready, we moved on with the ligation protocol. We stayed with an insert: backbone concentration ratio of 5:1 for all synthetic sequences and we left all three ligation reactions at 16°C for 16h, then we inactivated the ligase at 65°C for 20 minutes. This time no control ligation was done.
4. After the ligation was complete, we transformed our ligation products in chemical-competent Top10F’ bacteria: the external recombinant DNA entered the bacterial membrane thanks to a heat shock, performed by leaving the bacteria and the ligated DNA together at 42°C for 1 minute, then immediately transferring them on ice for 1 minute. After the heat shock was performed, we left our transformed bacteria in LB to recover and to express their selection marker gene, consisting in a kanamycin antibiotic. Afterwards, we plated the bacteria in LB agar with kanamycin and left them at 37°C until colonies started to grow.
5. When a decent number of colonies was present on all three plates, we picked all colonies that grew on them and performed a colony PCR.

   We performed this step in two different ways: at first we still used the original primers that were used as during the cloning of BBa\_K5109001, then we switched to a different set of primers that would match to the DNA a few bases outside the synthetic expression cassette and land on the pJUMP backbone, to obtain a more targeted binding and avoid any aspecific binding results. When the PCR reaction was completed, we ran all our results in gel electrophoresis to see if cloning was successful this time.

## CLONING OF EXTRACELLULAR LACCASE E

We were planning to create a new composite part for the expression of Laccase E found in E. coli’s genome thanks to our bioinformatic analysis. Due to time restrictions we ordered the extracellular expression cassette containing Laccase E’s coding sequence, hoping we would be able to clone it. However, due to issues to this composite part’s synthesis, we were not able to get it shipped and perform the ligation and digestion process with it.

## ENZYMATIC TESTS

In order to test the correct functioning of dehalogenases, we decided to set some tests that would use chloroacetic acid. Dehalogenases are able to break the bond that connects halogens to the carbon chain so, if the protein expressed by E. coli is correctly functioning, there would be the degradation of the molecule of the chloroacetic acid.

We tested 2 colonies (#4 and #9) of E. coli Top10F’ transformed with the Lpp-OmpT-DeHa2 construct by creating a medium composed of LB, chloroacetic acid 2.5 mM and IPTG 5 uM. We sampled the bacterial cultures after 0, 24 and 48 hours and treated the samples to lysate and remove the cells, in order to make them compatible
with the mass spectrometry analysis[2][3]. Every time point was treated with two different procedures, in order to determine both the concentration of chloroacetic acid in the medium and inside the cells and to be able to evaluate the internalization of the compound by E. coli.

The samples that we prepared were analyzed in the chemistry facility of our university.
Samples were thawed, diluted 1:100 with water/acetonitrile 50:50 and analyzed by FIA-HRMS (flow injection analysis - high resolution mass spectrometry). Briefly, 20 µL of sample were injected in the mass spectrometer (Q-Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer,
Thermo Fisher Scientific, Germany) by using acetonitrile as eluent at 0.300 mL/min flow.
Data acquisition was performed in negative ionization. The capillary voltage was set to 2.8kV, capillary temperature was 320°C, auxiliary gas and sheath gas were nitrogen (300°C) at 20 and 10 psi, respectively. Resolution was set to 35000, AGC target was 3x10 6 and max injection time was 200 ms. Full-scan range was within
m/z 50-250. Calibration was performed by Pierce ESI Negative Ion Calibration Solution (Thermo Fisher Scientific, Germany). The pseudo-chromatographic peaks of chloroacetate were identified and integrated by considering the Extracted Ion Chromatogram (EIC) of the [M-H] - species selected with a window of 5 ppm.

## GROWTH TESTS

#### E. coli in methanol

Methanol is an organic solvent that can be used for the desorption of GAC filters.
This can be done by using solutions of methanol 100% (24.7M) or ethanol 50% (8.56 M) with but this concentration is not compatible with the survival of E. coli. [4]
In the hypothesis where we use GAC filters to collect PFAS and we choose a solution with methanol
for the desorption, this solution would end up in the bioreactor with the bacteria. Both PFOA and Methanol are toxic so we decided to analyze firstly the toxicity of the two components alone and then together.
In order to have them together, representing the condition inside the bioreactor, we need to find a high concentration of methanol that allows the bacteria to grow and that can be used as a solvent for dissolving PFOA and PFBA in LB.
We decided to test these concentrations of methanol:
0,
0.25,
0.50,
0.75,
1.00,
1.25,
1.50,
1.75,
2.00,
2.25,
2.50,
2.75.

During the testing of E. coli growth, we learned to take some precautionary measures when working with methanol. In order to reduce the phenomenon of evaporation, we choose to prepare the stock solutions with methanol just before the preparation of the plate and, if necessary, put them in the fridge until that time.

#### E. coli in ethanol

Another option for the desorption of GAC filters, is to use a solution with ethanol. Similarly to what has been said with methanol, we decided to analyze how the growth is affected by increasing ethanol concentrations.
We used the same concentrations that were chosen for methanol.
Again, our goal is to find the concentration that allows the bacteria to grow and that can be used as a solvent for dissolving PFOA and PFBA in order to replicate the conditions inside the bioreactor.

Since ethanol has volatility problems too, we prepared our stock solutions just before the assembling of the plate.

#### E. coli in NaCl

Our project considers the use of anionic exchange resins, that would actually be a better candidate in our plant design, so there is the need to find a solution that can be used for their desorption. After a literature search, we found that one of the best desorbing solutions is water with NaCl 10%. [5]
E. coli is usually grown in LB which is an optimal cultural medium with 1% of NaCl. We decided to start from that concentration and increase it to 2%, 4%, 6%, 8% up until 10% and confront the results that are reported in the “results” page of the wiki.

#### E. coli in PFOA in LB

When working with PFAS, we decided to analyze increasing concentrations that will very likely be much higher than the one inside the bioreactor. This choice was done in order to understand what would be the maximum amount of PFAS that E. coli is able to endure.

We then decided to analyze PFOA and PFBA at these concentrations:
0 mM, 1.0 mM, 2.5 mM, 5.0 mM, 7.5 mM, 10.0 mM, 15.0 mM, 20.0 mM.

The choice to use LB as the solvent was done in order to have a better idea of the damage that is caused just by PFAS molecules to bacteria without any other variable.

#### E. coli in PFBA in LB

PFBA is a shorter molecule of PFAS that we decided to use after a conversation and visit “ACQUE VENETE”, a water distribution company in our territory. On this occasion, they told us that one of the most frequent types of PFAS that they found in waters was PFBA. We also hypothesized, would make it an easier molecule to degrade. For these reasons we decided to implement PFBA in our tests.Since there are not may studies about PFBA that test different concentrations, we decided to precede and test the same as we did with PFOA.

PFBA we used was in a liquid form, sensitive to light and also volatile so our experiments were done in a particularly controlled manner.

#### E. coli with PFOA and PFBA in methanol

The concentration of methanol that we chose for dissolving PFAS molecule was 1.00M. This choice was done after analyzing the growth rates and the final OD measured in the methanol experiments.

#### E. coli with PFOA and PFBA in ethanol

PFOA and PFBA were dissolved in ethanol 0.75M. This concentration was chosen after analyzing the growth rates and final OD in the ethanol tests.

#### E. coli with PFOA and PFBA with NaCl

According to literature, anionic exchange resins need a desorption solution with 10% of NaCl.
After the tests done with NaCl, we understood that the growth of E. coli would be almost completely inhibited after 2% NaCl. In order to replicate the condition inside the bioreactor,
we chose that concentration for more tests where the cultural medium contained both NaCl and PFOA and PFBA at increasing concentrations (the same as the ones in previous tests).
This type of experiment gave us a better idea of how E. coli reacts with those components and if a longer carbon chain as the one of PFOA would interact in a different way when compared with PFBA.

#### E. coli with chloroacetic acid

To test the correct functioning of the dehalogenase expressed on the surface of E.coli, we would need to test it with chloroacetic acid before trying with PFAS molecules.
For this reason, we were interested in knowing whether E. coli would be able to survive in a culture medium containing chloroacetic acid.
The concentrations of chloroacetic acid that we will use to test the function of the enzymes, will most likely be 0.5 mM and 2.5 mM so we were interested in testing the bacterial survival at these concentrations and a little beyond. We decided to choose concentration between 0 and 3.5 mM.

#### E. coli in synthetic wastewater

In the bioreactor, it is highly unlikely that LB will be the major component of the cultural medium for E. coli. Even though characterizing a process of E. coli growth using its optimal medium is a very effective way to understand the impact of the other molecules, working with synthetic wastewater
would give us a more realistic point of view. In order to do so, we decided to use a recipe that was previously used in a bioremediation research for the degradation of glyphosate [6]. Our first idea was , after creating the solution, to test if E. coli is able to survive with
the amount of nutrients that it contains and then replicate each growth test that we did using LB but substituting it with the synthetic wastewater.

This is the recipe that we firstly used for the synthetic wastewater:

* NaCl: 7 mg
* CaCl2: 3 mg
* MgSO4: 2 mg
* Beef extract: 100 mg
* NH4Cl: 199 mg
* Sodium acetate trihydrate: 163.6 mg
* K2HPO4: 18 mg

The results of this test are reported in the results page of the wiki and present the inability of E.
coli to grow in these conditions.
We then hypothesized that there weren’t enough nutrients present in the solution and tried to
modify the recipe by doubling the amount of carbon and nitrogen sources.
The synthetic wastewater recipe became the following:

* NaCl: 7 mg
* CaCl2: 3 mg
* MgSO4: 2 mg
* Meat extract: 200 mg
* NH4Cl: 398 mg
* Sodium acetate trihydrate: 327.2 mg
* K2HPO4: 18 mg

The results with the new recipe improved but the growth of E. coli in this condition was very far from the one
in LB and showed a lack of replicability.
For these reasons, we decided to scale up our experiment, hoping that larger volumes would help the bacterial
growth. To do this, we decided to change our experimental setup and, instead of using the plate reader, to
perform a growth test in shaking flasks.

In order to have more quantitative data than we had with the previous experiments, we decided to measure
the dry weight in addition to the OD600. These tests were done every two hours and we collected data
that has been plotted into graphs presented in the
[results](https://2024.igem.wiki/Uni-Padua-IT/results) part of the wiki.

#### E. coli in IPTG

The plasmid that has been used for the transformation of E. coli Top10F’ strain, enables bacteria to produce our desired protein, the dehalogenase DeHa2, under one condition: it needs to be inducted with IPTG.
Our previous attempts of cloning showed a significant toxicity of this protein in E. coli. Therefore, we were interested in knowing what the ideal concentration of IPTG that we could use for the induction would be and also have an insight on the toxicity of the dehalogenase on the bacteria itself.
For this reason, we decided to perform a test in which we analyzed the growth rates and the final ODs of the Top10F' strain that express the dehalogenases after induction with different concentrations of IPTG.
We choose to start from a very low concentration of inductor (0.5 µM) and increase it by a 10 factor until we reached the optimal concentration (0.5 mM).

Our tests were done at the plate reader that measures the OD 600 every 5 minutes for about 14 hours. The results that we obtained are reported in the
[results page](https://2024.igem.wiki/Uni-Padua-IT/results) of the wiki.

References:

*- Jacobus AP, Gross J (2015) Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli. PLoS ONE 10(3): e0119221. doi:10.1371/journal.pone.0119221
- Farajollahi S, Lombardo NV, Crenshaw MD, Guo HB, Doherty ME, Davison TR, et al. Defluorination of Organofluorine Compounds Using Dehalogenase Enzymes from Delftia acidovorans (D4B). ACS Omega. 2024;9(26):28546–55.
- Vascon F. A Synthetic Biology Approach to PFAS Bioremediation: Preliminary Computational and Molecular Studies on Fluoroacetate Dehalogenase [Tesi di Laurea Magistrale in Biotecnologie Industriali]. Università degli Studi di Padova; 2019.
- Deng S, Nie Y, Du Z, et al. Enhanced adsorption of perfluorooctane sulfonate and perfluorooctanoate by bamboo-derived granular activated carbon. Journal of Hazardous Materials. 2015;282:150-157. doi:10.1016/j.jhazmat.2014.03.045
- Dixit F, Barbeau B, Mostafavi SG, Mohseni M. Removal of legacy PFAS and other fluorotelomers: Optimized regeneration strategies in DOM-rich waters. Water Research. 2020;183:116098. doi:10.1016/j.watres.2020.116098
- Borella L, Novello G, Gasparotto M, et al. Design and experimental validation of an optimized microalgae-bacteria consortium for the bioremediation of glyphosate in continuous photobioreactors. Journal of Hazardous Materials. 2023;441:129921. doi:10.1016/j.jhazmat.2022.129921*

## Let's find out more about our project

### Project

[![Description Icon](https://static.igem.wiki/teams/5109/footer/description-icon.svg)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)
[Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)

[![Design Icon](https://static.igem.wiki/teams/5109/footer/design-icon.svg)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)
[Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)

### Wet Lab

[![Lab steps Icon](https://static.igem.wiki/teams/5109/footer/lab-steps-icon.svg)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)
[Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)

[![Results Icon](https://static.igem.wiki/teams/5109/footer/results-icon.svg)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)
[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

# {{ self.title() }}

{% block lead %}{% endblock %}

* Biology
* Sensor
* Filtration

* [Choice of enzymes](#Titolo0)
* [Clonings](#Titolo1)
* [Growth tests](#Titolo2)
* [Enzymatic tests](#Titolo3)
* [Future perspectives](#Titolo4)

# Choice of enzymes

We successfully managed to find all four candidates we needed: two dehalogenases and two laccases. Each enzymatic group is composed by one well characterised enzyme and one obtained through literature and bioinformatic analyses.

These enzymes needed to be tested to verify their effectiveness in PFAS bioremediation, to do so we planned to engineer different E. coli populations to express these enzymes on their surface.

![](https://static.igem.wiki/teams/5109/results/choice-of-the-enzymes1.png)

#### Dehalogenases

For the first dehalogenase, we decided to focus on one of the five dehalogenases from Delftia acidovorans, isolated by the USAFA iGEM 2020 team. Among the five dehalogenases isolated from D. acidovorans, only DeHa2 and DeHa4 demonstrated defluorination capability. Docking simulations were performed, and DeHa2 (WP\_011137954.1) achieved the highest score, making it our final choice.

For the second dehalogenase, we conducted multiple BLAST analyses, where UPI00000C10BF, an alpha/beta hydrolase fold enzyme from Synechocystis PCC 6803, emerged as the top candidate overall. Subsequent docking simulations yielded positive results, leading us to select this dehalogenase.

#### Laccases

For the first laccase, we selected a well-characterised E. coli laccase (P36649), which has been used in past iGEM projects for various bioremediation purposes, and we aimed to test its potential for PFAS bioremediation as well.

For the second laccase, we conducted several BLAST analyses, where MEB3229074.1, a multicopper oxidase from Synechocystis PCC 6803, emerged as the leading candidate. Further docking simulations showed promising results, leading us to choose this laccase.

# Clonings

#### pJUMP29-1A NdeI site mutagenesis

Mutagenic PCR was performed on our pJUMP29-1A original vector thanks to a primer pair mapping directly on the NdeI first restriction site.
To see whether mutagenic PCR was effective, the result from the mutagenic PCR was digested with 3 enzymes, EcoRI, PstI and NdeI: if three distinct bands at respectively 2kb, 700kb and 600kb were to be seen, it meant that the plasmid had not been mutagenized, while two distinct bands at 2,9 kb and 500kb meant that the restriction site was modified.

![](https://static.igem.wiki/teams/5109/results/cloningsresults1.jpg)

Columns 1,2,3 come from our mutagenesis result, and in columns number 2 and 3 we can clearly see two distinct bands at 2.9 and 0,5 kb: meaning that the mutagenic PCR was successful. Gel extraction was performed to proceed with other experiments.
For further reassurance, the mutated plasmid was sequenced, and this confirmed the correct mutation of the NdeI site: this is now a new improved part in the registry named pJUMP29-1A ∆NdeI (Part BBa\_K5109060).

This is the chosen backbone we used for further cloning experiments.

#### Cloning of DeHa2 intracellular expression cassette

Our intracellular expression cassette containing the DeHa2 coding sequence was amplified through PCR with our primers mapping on Prefix and Suffix, then digested with EcoRI and PstI and cloning and we attempted to insert it in our pJUMP29-1A ΔNdeI vector.
Bacteria were transformed with our ligation products and then plated on LB-kanamycin plates to select colonies containing our plasmid with the kanR gene.
Even though some colonies were able to grow on our kanamycin plates, everytime we performed either colony PCR or minipreps on inocula coming from these colonies we always found out that the vector they
contained was a result of partial digestion and subsequent self-ligation of the plasmid itself, always showing on electrophoresis gels bands at 400-500 bp corresponding to the original insert of the backbone.
Due to time limitations, the intracellular expression of [BBa\_K3347010](https://parts.igem.org/Part%3ABBa_K3347010) has yet to be achieved.

#### Homologous recombination

This procedure was performed as an alternative to the digestion-ligation protocol since no positive results came back from trying to clone our construct in pJUMP29-1A ΔNdeI.
After transforming our bacteria with the insert and vector mix with a 3:1 insert-backbone proportion, our bacteria were plated on an Lb+kanR plate, but it gave us a negative result as no colony grew on the plates. After this attempt, we went on with more traditional cloning attempts, since it was the cloning technique we knew best.

#### Cloning of extracellular DeHa2 and extracellular Dehalogenase S

Right after the idea of using our new primers with flanking sites at the extremities with Top10 F’ host cells, we decided to start working immediately on our extracellular expression cassettes by amplifying both DeHa2 and Dehalogenase S cassettes.
We performed digestion and ligation assays and then we transformed our Top10F' competent cells and plated them on LB kanamycin petri dishes.
On our first try, we could see that some colonies grew on both of our agar plates (Lpp-OmpT-DeHa2 plate on the top, Lpp-OmpA-Dehalogenase S plate on the bottom)

![](https://static.igem.wiki/teams/5109/results/cloningsresults2.jpg)

![](https://static.igem.wiki/teams/5109/results/cloningsresults3.jpg)

Given the small amount of time left before the wiki freeze, we decided to screen every colony available through colony PCR.

![](https://static.igem.wiki/teams/5109/results/clonigsresults4.png)

In colonies #4 and #9 a 1500 bp long band, corresponding to our synthetic sequence length, was visible, meaning that the expression cassette for the extracellular expression of DeHa2 was successfully cloned in pJUMP29-1A ΔNdeI in E. coli Top10F'.

Afterwards, we prepared our successful clones of [BBa\_K5109023](https://parts.igem.org/Part%3ABBa_K5109023) (expressing DeHa2) and sent them to the sequencing facility. Right before the wiki freeze, sequencing results came back, and we were able to confirm colony PCR’s results. Unfortunately, due to some technical problems, the quality of the sequencings we obtained was quite poor in some sections: therefore we could not determine if there were any mismatches or deletions in our colonies.

After the first colony PCR, we decided to repeat it for all the Lpp-OmpA-Dehalogenase S colonies that gave an inconclusive result. Colony #4 still gave us an inconclusive result.

![](https://static.igem.wiki/teams/5109/results/cloningsresults5.png)

We decided to send our uncertain clone #4 of BBa\_K5109020 (expressing Dehalogenase S) to the sequencing facility, just to be sure not to delete all possibilities of a successful cloning: surprisingly, this clone of BBa\_K5109020 also revealed a clear homology with our synthetic expression cassette, confirming that we obtained a successful cloning of the expression cassette for the extracellular expression of Dehalogenase S in pJUMP29-1A ΔNdeI in E. coli Top10F’.

#### CLONING OF EXTRACELLULAR LACCASE S

This cloning was done simultaneously to those of the synthetic sequences BBa\_K5109020 and BBa\_K5109023, but this sequence was not successfully cloned in Top10 F’. A reason to this failed attempt may lie on the significant length of BBa\_K5109022 (Laccase S expression cassette), being 2232 bp long, and the difficulties it may cause with the cloning of it in a vector not so much longer than the expression cassette (the pJUMP vector is 3374 bp long) and also the fact that, due to time limitations, we could not reiterate this cloning many times.

# Growth tests

Results of this part of the experiment are available in the PDF form down below.
[Growth tests-results.pdf](https://static.igem.wiki/teams/5109/results/growth-tests-results/growth-tests-results.pdf)

# Enzymatic tests

To verify the activity of DeHa2, we performed an assay of degradation of the chloroacetate substrate that can be converted into glycolate after the removal of the chlorine done by the dehalogenase.

The data obtained from the FIA-MS (flow injection analysis - high resolution mass spectrometry) measurements were analyzed using GraphPad Prism 10 and a Student’s t-test was conducted to determine if there was any significant difference between the measures taken at the different timepoints.

All samples grew in a medium containing chloroacetic acid 2.5 mM and were then differentiated between the methods used for the cellular lysis. For sample A we used osmotic lysis so the cultural medium was substituted with water while for sample B we used the french press maintaining the cultural medium.

After analyzing the data, the first observation we did was that the samples that contained only the cellular lysate (A) did not contain chloroacetic acid, since the concentration resulted lower than the limit of detection (LOD). This can allow us to say that the compound cannot be internalized by E. coli and remains in solution.

For what concerns the samples obtained after lysing the cells with the french press keeping the medium (B), we obtained the following results:

![](https://static.igem.wiki/teams/5109/results/enzymatic-tests/enzymatic-test1.png)

In this graph, we can see the variation of acid chloroacetic with time in the two different colonies transformed with the Lpp-OmpT-DeHa2 construct (colony #4 and #9) and in the wild type Top 10 F’ used as negative control. Both colonies were analyzed with and without the inductor for the expression of dehalogenase.

![](https://static.igem.wiki/teams/5109/results/enzymatic-tests/enzymatic-test2.png)

Percentage reduction in chloroacetic acid concentration

At first glance, we can see how colony #4 did not degrade the chloroacetic acid and gave similar results to the wild-type strain. On the other hand, colony #9 managed to degrade almost all the chloroacetic acid even when not induced. This may suggest a possible leaky activity of Ptac promoter.

To determine if there was a significant variation (and therefore some enzymatic activity) of the acid concentration in the samples, we conducted a Student’s t-test.

We calculated the value of t using the formula:

![](https://static.igem.wiki/teams/5109/results/result-formula1.webp)

, in which x is the mean of the measurements and s is the standard deviation, and the degrees of freedom with the formula:

![](https://static.igem.wiki/teams/5109/results/result-formula2.webp)

, in which n is the number of replicates and P the number of parameters evaluated (P=1).

![](https://static.igem.wiki/teams/5109/results/enzymatic-tests/enzymatic-test3.png)

In the table we reported the integrated chronogram peak areas for chloroacetic acid (I AcCl, obtained with FIA-MS and measured in relative abundance arbitrary units - aU) and the mean and the standard deviation for each group of samples.

The results obtained by the test are the followings:

![](https://static.igem.wiki/teams/5109/results/enzymatic-tests/enzymatic-test4.png)

The number of degrees of freedom was approximated by default.

No significant difference in the concentration of chloroacetic acid was measured at the different time points in either Top10F' wild type or colony #4 transformed with the Lpp-OmpT-DeHa2 construct.

There was instead a significant reduction in the concentration of chloroacetic acid in the colony #9 transformed with the Lpp-OmpT-DeHa2 construct. Specifically, a reduction of 97.94% was observed in the samples in which the expression inducer was present, compared to a reduction of 90.23% in the samples in which it was absent.

# Future perspectives

#### Clonings

We plan to try again with the cloning of BBa\_K5109022 (Laccase E expression cassette) as soon as possible, to have at least one laccase to have for our future enzymatic assays.

One of our goals is also to be able to clone the main intracellular expression cassette in pJUMP29-1A.
From this starting point, we would be able to switch enzyme sequences and create four different vectors expressing our protein of interest intracellularly.

#### Growth test

Since the growth test with PFOA in a medium containing 2% NaCl did not provide a clear correlation between the PFOA concentration and the inhibition of the growth, further investigation is required.

Clearly, we plan also to conduct other growth tests using the transformed bacteria, in order to see if there are any differences with the wild type, determine if the reaction occurs on our final target molecule and whether any toxic intermediates are produced.

#### Protein expression and enzymatic tests

From what can be seen in the section above, Lpp-OmpT-DeHa2 colony #9 showed the ability to process almost the entirety of chloroacetate dissolved in the culture medium, meanwhile colony #4 showed no sign of degradation activity.

Further investigation is necessary to understand why both of these phenomena are happening.

As mentioned before, we were able to get our sequencing results back just right before wiki freeze. They seemed to have a positive outcome for colony #9, while a deletion changing the reading frame is likely to be present in colony #4, but we did not have enough time to deeply investigate these results and we proceeded with the enzymatic tests regardless.

Regarding analysis on colony #9, we should perform SDS-PAGE and Western blot assay to verify the correct expression of our protein of interest.

These tests would be particularly useful to analyze superficial expression of DeHa2 fused together with the anchoring motif: thanks to our thrombin cut site situated between the C-terminal of truncated OmpT and the N-terminal of our enzymes, we can subject our engineered bacteria to thrombin treatment, thus allowing the release of our enzymes in the medium. Through subsequent cell precipitation through centrifugation and running the surnatant in SDS-PAGE, it could be possible to analyze the efficiency of our surface display system by measuring how much cleaved protein is detected.

Pelleted cells from previous centrifugation could also be lysed and cytoplasmic and membrane fraction could be separated and run on SDS page, thus verifying the presence of the anchoring domain in our membrane or formation of inclusion bodies.

Regarding enzymatic assays to verify our proteins’ degradation activities, there are a series of tests that we had planned to perform.

From what concerns DeHa2, we could further investigate its activity modifying the parameters that we used in our first test. Specifically, we could either increase the concentration of IPTG to try to obtain an increased protein production (above the conservative limit adopted due previously observed colony #4 IPTG-growth response), or lower it to assess the tunability of the system, and we could try to use closer time points to better characterize the kinetics of the reaction.

In addition to that, chloroacetate tests should be performed also on Dehalogenase S, meanwhile tests with reliable substrates should be performed on Laccase S and E.

By carrying out research in literature, we found syringaldazine as one of our possible candidates: syringaldazine is a substrate that changes its absorbance spectrum when oxidized by laccases, making degradation activity measurable with a spectrophotometer.

Lastly, degradation tests need to be conducted on the previously selected perfluoroalkyl substances - PFOA, PFOS and PFBA - on which we have performed several growth tests, to determine the feasibility of our system for PFAS degradation.

The previous one is the last step right before we introduce our four populations of engineered bacteria in a bioreactor and let the enzymes perform their catalytic activity synergically, which will hopefully lead to a total degradation of these pollutants.

We believe that such a biological approach needs an outline system that allows it to be utilized to the fullest. The system we have ideated is described in
[Plant Design - Overview](https://2024.igem.wiki/Uni-Padua-IT/overview).

* [Feedback & testing](#TitoloA1)
* [Future perspectives](#TitoloA2)

# Feedback & testing

After a few tests with different functionalization molecule designs (11-mercapto1-undecanol as functionalization molecule and NaCl as electrolyte), we managed to get the first results with GV38 as functionalization molecule and Hepes as electrolyte. More information about measurement cycles is shown on
[Engineering](https://2024.igem.wiki/Uni-Padua-IT/engineering).

The first round of measures with this functionalization got us signals with SERS and EIS as well. The graphs below are created by the union of three measurements, bare surface, functionalized surface and functionalized surface + PFOA solution.

The first two graphs show us how the Impedance module and phase changed between the three measurements.

![](https://static.igem.wiki/teams/5109/results/results-sensor/resultsensor1.jpg)

![](https://static.igem.wiki/teams/5109/results/results-sensor/resultssensor2.png)

The most interesting part of the graph is the range 70-1000 Hz, since with this range of frequency we can better appreciate the effects of the capacitance change in the system.

![](https://static.igem.wiki/teams/5109/results/results-sensor/resultssensor3.png)

As we can see, for higher values of frequency (higher than 1000Hz), we have differences between the measurements. These differences are mostly due to the resistance of the solution and the electrode. This parameter is hard to control, since the position of the electrodes and the evaporation of the solution can easily modify it.

The measure of functionalized surface + PFOA solution shown in the first graph was taken immediately after the PFOA solution was placed in our chamber. We have taken two other measurements at 30-minute intervals. The final measurements stated a stable interaction between the PFOA and the surface, since the impedance remained almost exactly the same.

![](https://static.igem.wiki/teams/5109/results/results-sensor/resultssensor4.png)

After carrying out the SERS analyses, we can confirm the presence of PFOA above the sensor. So the adhesion of the PFOA molecules to the surface is happening.

The PFOA powder's spectra show us this chemical compound's spectral characteristics, so we used it as a reference.

![](https://static.igem.wiki/teams/5109/results/results-sensor/resultssensor5.png)

Observing the PFOA on sensor analyses, we can see two peaks, respectively about the functionalization and the presence of carbon-fluorine bonds. This confirms the presence of PFOA on the functionalized surface of the sensor.

![](https://static.igem.wiki/teams/5109/results/results-sensor/resultssensor6.png)

We wanted to test our prototype with PFOS and PFBA, so we fabricated another set of sensors.
This round of testing has been performed due to technical problems with polypropylene chambers, which are not optimal for PFAS analysis because of their high tendency to adsorb them.

In the graphs, we can see the measurements of the functionalized surface (functionalized), then the measurements after the PFAS solution addition from T0(0min) to T3(120min) at 40-minute intervals.

With the PFOS we had no significant shifting in the impedance spectrum, while the PFBA seems to interact with the surface but with less accentuated shiftings than PFOA.

![](https://static.igem.wiki/teams/5109/results/results-sensor/resultssensor7.jpg)

![](https://static.igem.wiki/teams/5109/results/results-sensor/resultssensor8.jpg)

![](https://static.igem.wiki/teams/5109/results/results-sensor/resultssensor9.jpg)

![](https://static.igem.wiki/teams/5109/results/results-sensor/resultssensor10.jpg)

Further analysis of this data has been reported in the [modeling section](https://2024.igem.wiki/Uni-Padua-IT/model).

As a last experiment, we did a test with a new adjustment. We performed another EIS test, but this time we added in the solution the redox species ferry-ferrocyanide, hoping to enhance the effects observed in the past measurements.

![](https://static.igem.wiki/teams/5109/results/results-sensor/resultssensor11.png)

We managed to get some first promising results that will be further analysed on future occasions.

# Future perspectives

#### Implementation of other PFAS compounds

In our project, we focused on the detection of PFOA, the major pollutants of the PFAS family in Italy, as the analysis that Fredsense did with our samples revealed.

Having a sensor that works only on one compound can be useful even in industrial applications, for example, to assess if a filtering system is working correctly or in PFAS degradation processes.

A huge improvement would be to create a sensor that can detect multiple PFAS typologies.

In this case, there are two paths to reach the result:

* attracting multiple PFAS to the same surface, and thanks to the SERS properties, we are able to recognise them after the Raman analyses
* creating multiple surfaces able to interact only with one compound each in order to complete multiple EIS analysis

#### Groundwater analysis

Currently, we have only tested a simple solution where the possibility of interference from elements of the solution with our functionalized surface is really low.

A mandatory step before the commercialization of our sensor would be to determine the condition under which it can operate. The real solution will interface with our sensing system because there are many more elements dissolved.

#### Offline application

Once the characterization of our first prototype is completed and the applicability in real solution will be verified, we will be able to present our sensor to water facilities and other PFAS degradation companies.

Initially, it will be a disposable product

#### Surface regeneration

Based on the characteristics of the surface we are developing, we have good hopes of being able to make it reusable with proper regeneration.

The bond between the functionalization molecule and the surface proved out to be really strong, meanwhile, the forces that attract the PFAS to the surface are weaker.

This should make it possible for us to develop a regeneration process for our sensor in the future in order to cut the price even more.

#### Online application

This is the ultimate application for our project. Operating continuously in a water flow requires a lot of work and study. The surface needs to be able to attract and release PFAS in order to reach a certain equilibrium with the flowing solution.

This improvement would be a huge step for PFAS worldwide waters monitoring.

* [GAC regeneration](#TitoloB1)
* [AER adsorption](#TitoloB2)
* [AER regeneration](#TitoloB3)
* [Future perspectives](#TitoloB4)

# GAC regeneration

To analyze solvent regeneration of GAC we first looked at the scientific literature on the topic.
Through this phase and a collaboration with Professor Ester Marotta of the University of Padova
(for more information visit
[Collaboration & Partnership](https://2024.igem.wiki/Uni-Padua-IT/human-practices) - Expert in Organic Pollutant Degradation) we analyzed 2 possible organic solutions in 2 potential GAC mass - desorption volume ratio:

* MeOH : H2O + NaOH 0.1 M, 50:50 vs EtOH : H2O, 50:50
* 10 mg with 100 mL vs 200 g with 20 mL

The GACs used for the experiments were received from a water service provider in Veneto and were wet so we also compared those with a dried version of them.

1. Wet GAC vs Dry GAC
We found higher concentrations of PFBA and PFOS in the previously dried GAC (M1 vs M3 and M2 vs M4).
This was done by comparing the mass of PFAS found in the desorption solution, specifically comparing M1 with M3 and M2 with M4.

![](https://static.igem.wiki/teams/5109/results/filter-results/results1.png)

2. Selection of GAC mass - desorption volume ratio
To analyze the best GAC mass - desorption volume ratio we consider the latter as the variable parameter by considering PFAS concentration in the solution if we had the same amount of GAC.
In the comparison of the 2 possible ratios we found out that the use of higher volume for less activated carbon, so 100 mL for 10 mg, also presented higher concentrations (twice the amount of PFBA and 5 times for PFOS), as it can be easily expected. However to apply this kind of solvent regeneration in a real industrial application we do need to consider the substantial volume that this kind of ratio would mean: considering a total of 2000 kg of GAC with a desorption with ethanol and with a ratio of 200 mg - 20 mL we would need 100.000 L of ethanol whereas in the case of 10 mg - 100 mL we would need 10.000.000 L of just ethanol.

![](https://static.igem.wiki/teams/5109/results/filter-results/results2.png)

3. Selection of desorption solution
We compared the mass of PFAS found in the desorption solution, specifically comparing M3 with M4 and M5 with M6.

The solution with methanol and the one with ethanol gave similar results, for both ratio cases and for both PFOS and PFBA.

![](https://static.igem.wiki/teams/5109/results/filter-results/results3.png)

4. Desorption efficacy
The GACs tested were obtained in a real filtration system so we do not have data on the actual amount of PFAS present on the adsorbent prior to the desorption, thus it was not possible to determine the effectiveness of the desorption solution used. However, we did have the adsorption and desorption test results obtained by Prof. Marotta's doctoral student in which the solution with methanol and NaOH at a ratio of 200mg-20mL was used (corresponding to our M3 test); in this case the desorption efficacy was about 55% for PFBA and 59% for PFOA.
Using this notion we determined that the solution with ethanol with the same GAC-desorption solution ratio was about 53%.

Conclusions:
Through the data obtained from our GAC desorption experiments we understood that the solutions proposed have not the efficacy required to be applied to an industrial context. Moreover a scale-up of this chemical desorption technique would require elevated volume of methanol or ethanol, posing an environmental and economical issue (for the economical aspect visit
[Entrepreneurship](https://2024.igem.wiki/Uni-Padua-IT/entrepreneurship)).

# AER adsorption

The following table summarizes the results obtained from the analyses:

A860 - PFOA

![](https://static.igem.wiki/teams/5109/results/filter-results/filters4.png)

A110 - PFOA

![](https://static.igem.wiki/teams/5109/results/filter-results/filters5.png)

A111 - PFOA

![](https://static.igem.wiki/teams/5109/results/filter-results/a111pfoa.png)

A860 - PFBA

![](https://static.igem.wiki/teams/5109/results/filter-results/filters7.png)

A110 - PFBA

![](https://static.igem.wiki/teams/5109/results/filter-results/filters8.png)

A111 - PFBA

![](https://static.igem.wiki/teams/5109/results/filter-results/a111pfba.png)

However, from the data it can be seen that the best of the resins, in terms of adsorption kinetics, is A860.
We also see that all resins reach a plateau in PFBA adsorption. This behavior is similar to that of GACs, in fact we know from
meetings with Acuqevente that PFBA is the pollutant that first causes the filters they use to saturate.
In any case, A860 manages to adsorb more PFBA than the other resins, and is therefore the best of the three in terms of adsorption.

# AER regeneration

Regeneration in milliQ water
The following table summarizes the results obtained from the analyses:

![](https://static.igem.wiki/teams/5109/results/filter-results/tabfiltro.png)

In general, the regeneration of the resins did not bring noteworthy results.

The best appears to be A110 for PFOA and A860 for PFBA.

However, no surrender is suitable for our purposes.

In order to obtain valid alternatives to the GACs currently used, it is necessary to study chemical regeneration more thoroughly, carrying out more tests and also looking for new regeneration solutions.

#### Regeneration in tap water

The following table collects the results obtained:

![](https://static.igem.wiki/teams/5109/results/filter-results/tapwater.png)

The single regeneration value obtained again contrasts the data collected in the literature.

The efficiency of A860 it’s remarkable: although it was practically full from the first adsorption, it managed to re-adsorb almost all of the PFOA in solution.

We therefore think it is necessary to carry out further studies regarding the adsorption capacity of the resin and its regeneration..

In particular we suggest varying the volumes and concentrations of the solution.

We could also have varied these parameters, however we decided not to do so in an attempt to demonstrate the results of the article we used as a model. For our project it would be essential to achieve this objective as the ratio between the volume of resin used and the volume of the regeneration solution is extremely low (in particular the volume of the regeneration solution is equal to 10 times the volume of the resin). This characteristic translates from an ecological point of view into a lower use of water compared to GAC for example and from an economic point of view into a low cost due to chemical regeneration.

Other experiments could be performed with column regeneration tests, using a pressurized jet for example with a Buchner funnel.

#### Regeneration through concentration gradient

The resin analyzed adsorbed 9,64 \* 10^(-5), that is to say 96,4% of the PFOA in solution.
The following table reports the results obtained:

![](https://static.igem.wiki/teams/5109/results/filter-results/filters11.webp)

The regeneration rate was excellent: we in fact achieved a regeneration of 90,7% relative to an initial PFOA adsorption of 96,4%. However, we are not sure if this process is the most effective method to achieve such results, as several parameters were changed simultaneously: concentrations, exposure times, and volume of the regeneration solution used. Further studies are therefore required to optimize these parameters, particularly in anticipation of a scaling up of the process to an industrial level.

# Future perspectives

We believe that further studies should be conducted on GAC chemical desorption, analyzing other organic and non-organic solutions and focusing on their effect on Filtrasorb 400, as this type of GAC is the one used commercially and available to water service providers. In addition,
it is important to bear in mind the need to have low volumes of possible toxic solutions used in the scaleup system so as to be environmentally friendly and economically feasible.

The studied resins have performed very well in terms of filtering capacity and we believe that with a deeper regeneration characterization is needed.

We suppose that it’s necessary to confirm the results we found in the literature, then to try again to regenerate resins with different volumes or concentrations or even different solutions.

Other tests could be performed with a pressurized jet,

Through these experiments we’ll be able to demonstrate that anionic exchange resins are a valuable alternative to GACs.

## Let's find out more about our project

### Project

[![Description Icon](https://static.igem.wiki/teams/5109/footer/description-icon.svg)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)
[Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)

[![Design Icon](https://static.igem.wiki/teams/5109/footer/design-icon.svg)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)
[Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)

### Wet Lab

[![Lab steps Icon](https://static.igem.wiki/teams/5109/footer/lab-steps-icon.svg)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)
[Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)

[![Results Icon](https://static.igem.wiki/teams/5109/footer/results-icon.svg)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)
[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

{% endblock %}