There is a growing interest in emerging contaminants. They are compounds with several origins and chemical natures. Their presence in the environment, or the consequences of it, have not been fully glimpsed, causing environmental concerns and health risks.

In Mexico, few studies have determined the concentration levels of these compounds in wastewater, groundwater, and surface water, and almost all are made in the center of the country. According to Ronderos-Lara et al. (2020), among the reported compounds are erythromycin, estradiol, pentachlorophenol, bisphenol-A, naproxen, acetaminophen, diclofenac, rifampicin, and carbamazepine, among others. This situation is concerning if we consider the tremendous industrial and agricultural activity in a large part of the republic.

At Neotech-e, we aim to uncover a way to detect emerging contaminants using the power of synthetic biology. Hereafter, how our idea was developed is described in detail.

For the selection process of the emerging contaminants to be studied, we conducted an extensive review of technical articles on the presence of these compounds in the water bodies of our country. Within the established criteria, we decided to select three contaminants that appeared in a more significant number of technical articles and, if possible, reported a concentration in the body of water studied.

Finally, based on the review of the articles presented by Jacobo-Marín (2021), Arguello-Pérez et al. (2019), and Rocha-Gutiérrez et al. (2015), the emerging contaminants with the most significant presence in the Jalisco region, which delimits our community, are diclofenac, rifampicin, ibuprofen, ketorolac, pentachlorophenol, erythromycin, and estradiol. Of these seven compounds, it was observed that erythromycin and estradiol were found in concentrations that represent high toxicity in the effluents studied.

If experiments involving dilutions in water are desired, Shareef et al. (2006) report that estradiol is not soluble in water, so it is automatically discarded. The contaminants with a higher concentration reported were pentachlorophenol and rifampicin, followed by erythromycin. These three compounds are soluble in water, thus meeting the criteria for presence and solubility.

For instance, the three contaminants we chose to study are erythromycin, pentachlorophenol, and rifampicin.

To identify if the selected contaminants were present in the water bodies of our community, we decided to find enzymes that interacted with these contaminants. Nevertheless, we are not necessarily looking for enzymes that degrade our contaminant into something that can be uptaken by bacteria for a specific metabolic pathway, but the following criteria:

By performing a bioinformatic analysis, where we compared different enzymes from databases such as BRENDA, UniProt, and ProteinDataBank, among others, a table with different potential enzymes was elaborated:

Finally, the following enzymes were selected:

To test the functioning of each enzyme, we determined to register each sequence as a single BioBrick part. Nevertheless, the sequence reported previously is not going to be added as it is, but with the following modifications:

For instance, the scheme of our single BioBrick part is shown as follows:

With the rise of synthetic biology, biosensors have gained popularity over the years. Biosensors are devices that detect the presence of certain compounds by using living organisms or biological molecules.

Considering the idea of using a biosensor, we propose an enzymatic system based on how the Förster resonance energy transfer (FRET) operates as described by Carmona et al. (2006): one enzyme capable of recognizing and degrading a specific contaminant will be flanked by two fluorescent proteins. A fluorescent protein can be excited at a distinctive wavelength by a laser and emit an intensity absorbed by a detector. Based on this precept, energy transfer can occur as one of the fluorescent proteins, the donor, is excited and transmits energy for other fluorescent proteins, the acceptor, to emit.

The emission intensity of the acceptor can help us quantify the concentration of a ligand binding to a protein. For instance, we suggest a procedure capable of producing measurable fluorescence by interacting with a ligand with a characteristic enzyme, which correlates directly with the ligand concentration, showing us that the contaminant is either present or not in a sample.

Fluorescent proteins are most commonly used as donor and acceptor fluorophores in FRET biosensors, especially since these proteins are genetically encodable and live-cell compatible. For this section, we relied on the articles from Bajar et al. (2016) and Agrawal et al. (2021), where different fluorescent proteins are compared according to the requirements of a particular system.

The particularity of fluorescent proteins depends on three main advantages: fluorescent proteins-based biosensors are easily constructed via genetic engineering, they confer high cellular specificity by using specific promoters, and these systems are stable in cells for a long time due to high intracellular stability.

The first fluorescent protein pair developed was formed of enhanced blue fluorescent proteins (EBFP) and enhanced green fluorescent proteins (EGFP). However, its low brightness and low photostability made this pair impractical in most applications. Nowadays, cyan and yellow fluorescent proteins are used to overcome these difficulties.

From the pairs suggested by Bajar et al. (2016), enhanced cyan fluorescent protein (ECFP) and mVENUS (YFP) are widely recommended because of a higher quantum yield and better folding at 37 °C. This fact is also confirmed by Agrawal et al. (2021), who successfully developed a functional FRET-based sensor to monitor silver ions using this pair of fluorescent proteins. Agrawal et al. (2021) mention that the emission spectrum was recorded after excitation of the sensor protein at 420 nm, and recording the emission in the range of 450 to 600 nm, reaching a peak in 530 nm.

Sequences from both fluorescent proteins were obtained from the BioBricks catalog provided by iGEM, selecting these two BioBricks after analysis:

For the design of this project, each fluorescent protein is attached to one of our desired enzymes: EryK, PCP, and RifMO. Initially, the Enhanced Cyan Fluorescent Protein (ECFP) coding sequence is attached before our desired enzyme's start codon. In contrast, mVENUS is attached right after the last amino acid before the stop codon of our desired enzyme.

Nevertheless, we decided to add the HindIII restriction site between the Enhanced Cyan Fluorescent Protein (ECFP) and our enzyme's start codon, as well as the NdeI restriction site between the enzyme of our interest and mVENUS, to ensure other teams can use this system based on the previously described fluorescent proteins by changing the enzyme we use for any of their interest. The structure of our new composite part is displayed as follows:

Expression systems are genetic constructs designed to produce a protein, or an RNA, either inside or outside a cell. For our parts to be tested, it is necessary to build an expression system based on iGEM's Registry of Standard Biological Parts.

Expression systems are genetic constructs designed to produce a protein, or an RNA, either inside or outside a cell. For our parts to be tested, it is necessary to build an expression system based on iGEM's Registry of Standard Biological Parts.

First, we designed our system for a bacterial platform, selecting the Escherichia coli TOP10 strain for high-efficiency cloning and plasmid propagation and the Escherichia coli BL21 strain for suitable protein expression. Moreover, we determined to use pSB1C3 as the plasmid backbone, which propagates the BioBrick part and is widely used by other iGEM teams.

For instance, our expression platform is composed of the following elements: an inducible promoter, which is regulated in response to specific stimuli; a ribosome-binding site to bind the ribosome for the initiation of translation; and a transcription terminator that mediates the release of the transcript RNA from the translational complex. Using the BioBricks catalog provided by iGEM and considering the results delivered by each part entry, we concluded the following parts were the most optimal to use for our composite part:

To build our composite part, we decided the best option was to synthesize it with one of the iGEM sponsorships, Integrated DNA Technologies or Twist Bioscience. Nevertheless, considering the possible length of our composite part and the cost of getting erythromycin, pentachlorophenol, and rifampicin, we determined it was better to perform enzymatic assays with just one contaminant. For instance, erythromycin was selected because of its outstanding presence in our community's water bodies and the capacity to get a stock of it.

Finally, our composite part considering EryK as the enzyme that will interact with erythromycin is shown as follows:

To initiate testing each of the selected enzymes, we decided the best idea was to synthesize each gene and evaluate enzyme activity. To accomplish this, we ordered the genetic material corresponding to EryK, Pcp, and RifMo. In the case of Pcp and RifMo, we synthesized single genes, while for EryK, we ordered the whole FRET system, meaning we had to isolate EryK through PCR amplification.

When these genes arrived, the backbone plasmid in which our interested genes came was pUC57, containing the restriction sites we designed for future experiments. For instance, we performed a DNA restriction digest with NcoI and XhoI restriction enzymes to separate the desired system and our expression plasmid, pBAD/Myc-HisB. Up to now, we have successfully cloned EryK and the entire FRET system with EryK in pBAD/Myc-HisB.

For our bacterial expression system (BBa_K4447005), we ordered it with one of the iGEM sponsors. To do this, we had to split the entire system into four parts and, to join them, we added HindIII and NdeI restriction sites in 5' and 3' ends, respectively.

As mentioned before, we are using pBAD/Myc-HisB in Escherichia coli BL21 because of the edges this vector delivers: this plasmid is designed for regulated, dose-dependent recombinant protein expression and purification. Nevertheless, before starting with protein expression, we cloned our genes in Escherichia coli TOP10, ideal for high efficiency cloning and plasmid propagation. For our bacterial expression system, we opted to clone it on pSB1C3 because of the small number of base pairs and the fact that we already have a suitable promoter, ribosome-binding site, and terminator.

To screen for correct colonies, we designed a three-step verification:

During this phase, we suffered from several troubles that did not allow us to perform all of the expected experiments. First, we received an incomplete iGEM Distribution Kit with fewer plates than we were supposed to acquire. This fact represented a limiting step in our assembly phase because we were supposed to assemble fluorescent proteins with Pcp and RifMo using the parts provided by this kit. Also, parts we synthesized with iGEM sponsors have not arrived, even though they were ordered by the end of July, with the expected arrival schedule of one week after ordering. For instance, we have not been able to characterize our bacterial expression system (BBa_K4447005).

The first experimental step of the project was to clone the genes of interest: Pcp (BBa_K4447002), RifMo (BBa_K4447003), and EryK in the FRET system (BBa_K4447003). For this, the Escherichia coli TOP10 strain was transformed with the plasmid containing the gene of interest. The next step was to perform a miniprep to extract our plasmid: since our gene was in a maintenance plasmid such as pUC57, we performed restriction digest with XhoI and NcoI restriction enzymes to make a ligation in pBAD/HisB. Afterward, we repeated the process of transforming in the Escherichia coli TOP10 strain, but now our gene was located in pBAD/HisB. Next, a plasmid DNA extraction and a colony PCR were performed to ensure a correct transformation and eliminate colonies with self-ligations, to keep only the colonies that contain the whole construct.

The subsequent experimental step was to perform a bacterial transformation in the Escherichia coli BL21 strain to start with protein overexpression in the case of EryK. After this step, we picked the most isolated colonies from a Petri dish and grew them in liquid culture with carbenicillin, inducing them with L-arabinose. Next, we evaluated which cultures had higher turbidity and performed cellular lysis through sonication. To check if the desired protein was correctly expressed, we used the SDS-PAGE gel protocol described in our logbook, in which we analyzed lysed-induced cells. In addition to examining the induced colonies, we also analyzed a control to see differences in proteomic expression between induced and uninduced colonies to verify that the overexpression protocol was correct and effective.

The next step would be to measure the enzyme activity for EryK. For the moment, we are measuring it with a coupled system. This system works using the NADP+ produced by our enzymes that detect the drug, then we add an enzyme that oxidizes the NADPH to reduce another substance that depending on its hue and absorbance, helps us to determine the production of NADPH, thus indirectly giving us the concentration of erythromycin. On the other hand, the FRET system works with two components, the acceptor and the donor, where theoretically, every time our enzyme works, the system will be activated and generate luminosity, facilitating the detection of the drugs.