Parts

Introduction

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During our project, we designed, engineered, assembled, and tested many standard biological parts. Below you will find the description of our Best Composite Part (Special price) and an ingeniously designed collection to increase the specificity of our biosensor. You will also find the name, ID, and link to all the parts our team added to the registry, as well as the pre-existing parts we used.

Best composite part

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The gene the mqsA gene (b3021) in E. coli MG1566 has been identified by us as significantly up regulated under 100 µM PFOA 100 µM exposure (Wintenberg et al., 2025). In order to design a biosensor, the promoter of this gene serves as an entry point to link PFOA concentration to a measurable biological response, by using a reporter gene that translates the level of expression (which increases in the presence of PFOA) into an easily detectable signal, such as fluorescence.

To achieve this, we designed a simple construct (BBa_25O14TR6) consisting of:

• the mqsA promoter, identified by analyzing the read count table from Wintenberg et al. (2025) (BBa_25WX0GI9),
• a ribosome binding site (RBS) (BBa_K3715082),
• a red fluorescent protein, mRFP1 (BBa_K4822996),
• and a transcriptional terminator, the rrnB T1 terminator (BBa_K4800001).

This construct was used to transform strains closely related to those employed by Wintenberg et al. (2025) for RNA-seq experiments in E. coli exposed to 100 µM PFOA.

After a time-course experiment in which the transformed E. coli MG1655 ΔRM containing the construct was exposed to different concentrations of PFOA, we identified a time point (t = 24h) at which the relationship between RFP fluorescence normalized by OD600 (RFP fluorescence/OD) and PFOA concentration was optimal, over a range of 1.28 µM to 20 mM.

This construct was used to transform strains closely related to those employed by Wintenberg et al. (2025) for RNA-seq experiments in E. coli exposed to 100 µM PFOA.

The data suggest a potential proportional relationship between PFOA concentration and the RFP fluorescence/OD signal at 24 hours for strain b3021. To predict PFOA concentration from the fluorescence/OD value, we plotted PFOA (the variable to be predicted) on the Y-axis and fluorescence/OD (the predictor variable) on the X-axis, as regression models optimize the sum of squared residuals along the vertical axis.

Using a second-degree polynomial regression, we obtained a strong correlation between fluorescence/OD and PFOA concentration, modeled by the following equation:

[PFOA] = 1.63 × 10⁻⁴ × (RFP Fluo/OD)² − 1.92 × (RFP Fluo/OD) + 5443.9

From this model, it is possible to estimate the PFOA concentration within a 95% confidence interval using only the fluorescence/OD value at t = 24h for this strain (Figure 3).

This approach represents, to our knowledge, the very first attempt at enabling biological detection and approximate quantification of PFOA concentrations. Such a completely novel strategy could be applied for preliminary water screening, allowing rapid, low cost and widespread monitoring across numerous sampling points.

The collection part we designed in order to enhance the biosensor specificity

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The problem with a biosensor system that relies solely on the activation of a single promoter is that many other compounds may activate this promoter even in the absence of PFOA, leading to false positives. This can be observed in the last subsection of the “Result Detection” tab, where our construct containing the composite part does not provide reliable information about PFOA concentration when diluted in a more complex matrix, namely environmental water.

To address this issue and add robustness to the detection system, we envisioned a design in which a signal appears only if two promoters are activated simultaneously. In this system, two promoters respectively regulate two parts of the well-studied luciferase operon previously explored by the iGEM FiAtLux 2022 team. The genes responsible for synthesizing the luciferase substrate (luxC–luxD–luxE) are placed under the control of the first promoter, while the genes encoding the luciferase enzyme itself (luxA–luxB) are placed under the control of the second promoter. In this way, a luminescent signal is produced if and only if both promoters are active; if one promoter is activated without the other, no signal is generated (Figure 4).

In addition, we included a fluorescent protein in each translation unit to provide an internal readout of the activation level of each promoter. We tested this design using two inducible promoters (we did not have time to test with PFOA-sensitive promoters identified in our analysis) with the promoters pLac and pTet (Figure 5).

The collection part contained:

• Transcriptional terminator rrnB T1: BBa_K4800001
• luxB: BBa_K4239004
• luxA: BBa_K4239003
• eGFP: BBa_K1911005
• LacO operator to control the expression of the synthetic operon luxA-luxB-eGFP (BBa_K5178011) under the trc promoter, through LacI inhibition
• trc promoter (a variant of pLac): BBa_K4800000
• Terminator of LacI transcription
• LacI promoter and LacI: BBa_K5436200
• Terminator of TetR transcription: BBa_B1006
• TetR: BBa_K4818022
• Overlapping promoter that initiates transcription of both TetR and the synthetic operon containing mCherry, luxC, luxD, luxE: BBa_K4818023
• Tet operator, where TetR binds: BBa_K4818024
• mCherry: BBa_2583P08G
• luxC: BBa_K4239001
• luxD: BBa_K4239002
• luxE: BBa_K4239005
• Lambda t0 terminator: BBa_K4818021

We performed a time-course experiment under several conditions in quadruplicate: induction of only one promoter (first one or the other), induction of both promoters simultaneously, and no induction (Figure 6).

Normalized luminescence signal in E. coli DH5α transformed with either the collection part or the empty plasmid, under different induction conditions: single induction with 10 ng/mL aTc or 20 µM IPTG, double induction with 20 µM IPTG and 10 ng/mL aTc, or without induction.

* : p < 0,05 between the condition “Induction 20µM IPTG + 10 ng/mL aTc” and “non induction”,

** : p < 0,01 for the same conditions,

# : p < 0,05 between the condition “Induction 20µM IPTG + 10 ng/mL aTc” and “Induction 10ng/mL aTc”,

## : p < 0,01 for the same conditions,

† : p < 0,05 between the conditions “Induction 10ng/mL aTc” and “empty plasmid”,

†† : p < 0,01 for the same conditions.

Induction with IPTG alone did not result in a fluorescence level different from the baseline, whereas dual induction produced a significantly stronger signal, exactly as predicted by the design.

Induction with anhydrotetracycline (aTc) alone was less informative, since we observed that the pLac promoter exhibited substantial leakiness. As a result, the condition intended to represent single induction with anhydrotetracycline effectively became a dual-induction condition, because pLac was constitutively active. Despite this leakiness, we still observed a markedly stronger response under dual induction compared with single induction using anhydrotetracycline, supporting the increased robustness of this design.

Overall, these results confirm that this part collection favors specificity through simultaneous activation of both promoters, while also underlining the influence of pLac leakage and the need for further optimization.

This construct design emerges as a powerful template for enhancing signal specificity, with potential applications across a wide range of systems. While the constitutive leakiness of one promoter poses challenges and calls for further refinement, the overall architecture achieves a dramatic improvement in generating a uniquely specific response, demonstrating the promise of this strategy for precise synthetic control.

We had planned to apply this system using PFOA-sensitive promoters, in order to increase the specificity of the signal, which can be difficult to achieve with a single promoter in a complex matrix. For this approach to be effective, two promoters must be significantly activated at the same time point in the presence of PFOA. Our analysis of the study by Wintenberg et al. (2025) allowed us to identify two such promoters: BBa_257DQD3R and BBa_25WX0GI9, both regulating genes that were significantly upregulated in response to PFOA at t = 24 h.

List of all of our parts

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In this section, we present all the parts that our team added to the registry.

New parts

The two first tables contain the new parts for both the degradation protein and the promoter.