Results: Achievements of the Hydro Guardian
Welcome to the Results page of the Hydro Guardian. Explore the data, findings, and insights that demonstrate the effectiveness of our innovative biosensor and the impact we've made this year.
Welcome to the Results page of the Hydro Guardian. Explore the data, findings, and insights that demonstrate the effectiveness of our innovative biosensor and the impact we've made this year.
To develop our cell-based sensor for early and sensitive detection of antibiotic residues in wastewater, we designed various genetic constructs and transfected cells with three plasmids designed by us. The plasmids coding for a transmembrane protein, transcription factors and a reporter fluorophore driven by a specifically designed promoter are composed of different individual parts and were validated in several steps.
After cloning success was verified by PCR and subsequent gel electrophoresis as well as sequencing, our first set of cell culture experiments aimed for the validation of the functionality of the individual plasmids we designed. In the second step, co-transfection of multiple plasmids into HEK293T cells and subsequent analysis of reporter activity upon challenge with ampicillin was investigated.
Prior to the transfection experiments, the sequences for PknB (BBa_K5317013), CcpA (BBa_K5317014), GraR (BBa_K5317015) and ATF2 (BBa_K5317016) were inserted into the plasmid-backbone pEGFP-C2 (BBa_K3338020). For validation of localization and fluorophore intensity, fluorescent proteins like mRuby2 (BBa_K5317001) and EGFP (BBa_K3338006) were used. Via HiFi DNA Assembly, our designed composite parts were integrated into one plasmid each, which were introduced into E. coli DH5-alpha via heat shock transformation in the following. The correct transformation of competent bacteria was validated by colony PCR, followed by an expansion of the respective bacterial clones in liquid culture, enabling plasmid isolation and conclusive sanger sequencing. The ready-to-use composite parts for our antibiotic biosensor are shown in the following Figures 1 to 4, which present a schematic plasmid feature map as well as gel electrophoresis images of PCR amplicons obtained for cloning validation purposes.
The successful construction of the in silico-designed composite parts is illustrated in Figures 1 to 4. These include CMV-EGFP-PknB (BBa_K5317018), CMV-CcpA-mRuby2 (BBa_K5317019), CMV-GraR-mRuby2 (BBa_K5317020), and CMV-ATF2-mRuby2 (BBa_K5317021). The correct introduction of our constructs was shown via analytic PCR for the clones five and nine of BBa_K5317018 (2229 base pairs) and for clone 10 of BBa_K5317021 (3444 bp). The correct introduction of the constructs BBa_K5317019 (1977 bp) and BBa_K5317020 (1665 bp) could be shown via Colony PCR. A further validation via sequencing was conducted for all plasmids to verify no mutations were inserted into the sequence during the cloning procedure.
The ATF2-responsive promoter 3xCre3xAP1-miniCMV-miRFP670 was designed in the second cycle of the engineering circle. The in silico-constructed part 3xCre3xAP1-miniCMV promoter (BBa_K5317017), and the part miRFP670 (BBa_K5317002) were introduced in the backbone vector pEGFP-C2 via HiFi DNA Assembly, followed by heat shock transformation of the assembled plasmid into E. coli DH5-alpha.
The successful construction of the composite part ATF2-3xCre3xAP1-Promoter_miniCMV_miRFP670 is shown in figure 5. Here, you can see that the composite part ATF2-3xCre3xAP1-Promoter_miniCMV_miRFP670 is replacing the CMV-promoter and EGFP sequence originally present in the plasmid backbone. The correct introduction of our construct was shown for the clone seven via colony PCR, where a band with the expected sizeof 1159 bp was detectable. A further validation via sequencing was performed to verify no mutations were introduced into the sequence.
As shown in Figure 6, EGFP-PknB is correctly expressed in HEK293T cells. Very delightful is the membranous installation of the codon-optimized prokaryotic membrane protein in the eukaryotic cell membrane. The presence of the protein in the cells has been a very important and challenging part of our work, as the PASTA domain of PknB is crucial for β-lactam detection in the extracellular medium. With this, we were able to continue to find a functional transcription factor for signal transfer in the cell.
Within the first round of transfections, we discovered no or a very weak expression of our composite parts Ccpa-mRuby2 and GraR-mRuby2. As a result of this, we decided not to use these constructs for the co-transfection with EGFP-PknB. The correct expression of ATF2-mRuby2 is shown in Figure 7, where a cytoplasmatic localization in the cells is detectable. Therefore, we decided to use ATF2 as a crucial transfection factor to mediate between the PknB and the promoter. However, it is important to note that only small sample sections are presented here, and transfection efficiency may vary between treatments.
The co-transfection of the functional EGFP-PknB and ATF2-mRuby2 is shown in Figure 8. The expression of both parts was detectable, which are also located in one cell. This enables us to plan in a further step a promoter, which will be activated by ATF2.
Here, Figure 9 shows HEK cells co-transfected with our composite parts EGFP-PknB and ATF2-mRuby2 as well as our tested promoter-driven reporter. ATF2 expression is shown in red and the respective reporter miRFP670 expression, which indicated a basal promoter activity, in pink. Particularly noteworthy here is again the correct localization of the prokaryotic membrane protein PknB in the eukaryotic cell membrane, in green. As a human transcription factor, ATF2 plays a crucial role in DNA-binding to regulate gene expression Text. A basal activity of our promoter could therefore be due to possible ATF2-activity in the HEK cells that could interact with the promoter even without further antibiotic stimulus from the environment (Figure 9a).
To study whether the presence of β-lactam antibiotics in the cell media will be sensed by PknB, leading to a phosphorylation of ATF2 and subsequently to an induction of our promoter-driven reporter fluorophore, we incubated co-transfected HEK293T cells with ampicillin (100 µg/mL) for four hours.
The change of the miRFP670 reporter’s fluorescence intensity upon challenge was microscopically analyzed and is captured in the Figure 9b. Here, a qualitative change could be shown, which indicates a functional PASTA domain activity followed by ATF2 phosphorylation leading to miRFP670 expression. To address whether this change in reporter fluorescence intensity can also be aquired in a quantitative manner, we performed a flow cytometry analysis.
The qualitative change in the fluorescence signal was finally determined quantitatively using FACS analysis. The results are presented in the bar chart displayed in Figure 10. Here, the percentage of cells expressing the fluorophore under the control of the tested 3xCre3xAP1-miniCMV promoter is displayed as a function of various concentrations of ampicillin. A general increase across the various concentration levels can be seen after four hours of incubation, with the greatest increase from the basal level to 2.5 µg/mL. As a result, a saturation can be recognized for higher concentrations. Therefore, it can also be said here that a successful detection of ampicillin by our sensor is possible, but a closer look at concentrations between 0 µg/mL and 2.5 µg/mL will be interesting to study in future experiments.
In addition to β-lactam antibiotic detection, we designed an additional innovative cellular sensor for the detection of heavy metal residues in wastewater, such as copper, zinc and cadmium. To do this, we developed various genetic constructs and transfected cells with two self-designed plasmids. One of the plasmids contains the Metal-responsive Transcription Factor-1 (MTF-1), the other the associated MRE-containing promoter.
MTF-1 (BBa_K5317007), as a nucleocytoplasmic shuttle protein, plays a crucial role in the recognition and cellular response to heavy metals. When heavy metals are present in the cell, it accumulates in the cell nucleus, where it binds to promoters containing a Metal-Responsive-Element (MRE) and thus specifically regulates gene expression Text. For our MRE-containing promoters, we designed in silico four different basic and composite parts. We used the wildtype (BBa_K5317003) sequence of the metallothionein promoter Text as one version and for the others 4x the sequences “a” (BBa_K5317004), “d” (BBa_K5317005) or the combination of both “dada” (BBa_K5317006). As an example, we show the ready-to-use plasmid of the MREwt promoter (Figure 12).
The successful construction of the composite part CMV-MTF1-mRuby2 (BBa_K5317012) is shown in Figure 11. Here, you can see that CMV-MTF1-mRuby2 is replacing the eGFP sequence within the plasmid backbone. The correct assembly of our construct was shown for the clone five and eight via colony PCR, where a band with the expected size of 3019 bp was detectable. A further validation via sequencing was conducted and verified no mutations were inserted within the sequence during cloning.
The successful assembly of the composite parts MREa-EGFP (BBa_K5317009), MREd-EGFP (BBa_K5317010), MREdada-EGFP (BBa_K5317011), validated via PCR analysis, are shown in Figure 12, where also a representative plasmid feature map of MREwt-EGFP (BBa_K5317008) is displayed. As can be seen, MREwt-EGFP was integrated into the plasmid backbone replacing the originally present CMV-promoter, thereby driving the expression of the eGFP reporter. The correct assembly of our constructs was shown for all MRE-containing promoters by PCR analysis yielding a band at 230 bp on the agarose gel. A further validation via sequencing was performed for all plasmids, which verified sequence correctness.
In Figure 13, the expression of MTF1-mRuby2 in HEK293T cells is illustrated in red. The localization in the cell nucleus should be emphasized here. MTF-1 plays an important role for our metal biosensor, as it recognizes and binds heavy metals such as cadmium, zinc or copper in the cytoplasm Text. This leads to activation and binding to the promoter.
Moreover, a basal activity of the MRE-containing promoters without co-transfected MTF-1 were examined. These experiments showed no fluorescent signals, which indicates that there is no induction and that MTF-1 is probably necessary for promoter activity. With this information, we were able to continue with co-transfections of both parts.
In the second round, we co-transfected HEK cells with our MTF1-mRuby2 composite part and our four tested promoter-driven reporters. MTF-1 expression is shown in red and the respective reporter eGFP expression, which indicated promoter activity, in green (Figure 14a-d). We observed, that MTF-1 was continuously localized within the nucleus, while the reporter fluorophore showed a cytoplasmic localization, both as expected. A basal expression of the promoter-driven reporter fluorophore can be seen for all four promoters in case of no metal stimulation. This is due to possible metal ions in the culture medium of the HEK cells that could interact with the MTF-1. To evaluate whether the reporter fluorescence can be induced in case of the presence of metals in the cellular environment, we relied on microscopic analysis as a first qualitative approach.
After successfully localizing and expressing the composite parts, we conducted a further qualitative evaluation of reporter signal intensity in response to 500 µM copper sulfate. The fluorescence signals for the MREwt (Figure 15a, b) and MREdada (Figure 15c, d) promoters together with MTF-1 were observed before and after four hours of stimulation. Unfortunately, no clear visual increase in fluorescence was detected for these promoters compared to the baseline signal. However, it is important to note that only small sample sections are presented here, and transfection efficiency may vary between treatments. Visual assessment of fluorescence changes is challenging, which is why flow cell cytometry was subsequently considered to obtain a quantitative analysis.
The change in fluorescence signal was quantitatively assessed using FACS analysis. The results are presented in the bar chart in Figure 16. The percentage of cells expressing the fluorophore under the control of the tested promoter is displayed as a function of various concentrations of copper sulfate across all four promoters. It is evident that the MREa and MREdada promoters show an increase in activity, while the MREwt promoter exhibits no significant change, and MREd even shows a decrease in promoter activity upon stimulation. Notably, the MREdada-containing promoter exhibited the highest increase in fluorescence signal. Overall, our synthetic promoters, MREa and MREdada, enable effective detection of copper sulfate compared to the wild-type promoter.
We successfully designed biosensors for detecting both metals and β-lactam antibiotics.
For antibiotic detection, we introduced PknB, ATF2, and a promoter-driven reporter into HEK cells. Although challenging, we achieved successful localization of the prokaryotic membrane protein PknB in a eukaryotic system. Using the ATF2-sensitive 3xCre3xAP1-miniCMV promoter (BBa_K5317017), we demonstrated sensitive detection of up to 2.5 µg/mL ampicillin, both qualitatively and quantitatively. For higher concentrations, a saturation in fluorescent signal was measurable.
For metal detection, we integrated the transcription factor MTF-1 and four types of MRE-containing promoters. The MREa-EGFP and MREdada-EGFP constructs showed improved fluorescent reporter expression compared to the MREwt promoter, with detection up to 500 µM copper sulfate.
Despite these successes, fine-tuning is still needed for both detection systems, which we plan to address in future experiments.
