An accurate and sensitive copper biosensor is essential for the detection and monitoring of copper ions. While yeast biosensors have been developed for detecting heavy metal Cu²⁺, the existing yeast sensors for Cu²⁺ rely on the CUP1 promoter, which exhibits relatively high background noise. In order to achieve a biosensor with both low basal expression levels and high sensitivity, our goal is to design a synthetic circuit based on the yeast MAPK pathway to amplify the signals generated by the biosensor. Firstly, we control the expression of α-pheromone through the CUP1 promoter in response to Cu²⁺, allowing for fine-tuning of communication and response based on the concentration of Cu²⁺. Secondly, we introduce a positive feedback module to amplify the signals, thereby increasing sensor sensitivity and reducing the detection limit. Additionally, we have engineered the pheromone-inducible promoter to expand its dynamic range, achieving enhanced signal amplification effects. In the future, our aim is to modify the CUP1 promoter to lower basal expression levels and further enhance sensitivity, thus maximizing the impact of this innovative biosensor in the field of copper ion detection.

Figure 1-1 Concept map of design ideas

Design

The current methods for detecting copper ions are mainly divided into physical and chemical approaches, which offer relatively accurate and rapid detection. However, these methods often suffer from drawbacks such as high equipment costs and complex pre-processing procedures. Our team aims to develop a biological approach that enables convenient and sensitive detection of copper ions.

Figure 1-2 Design of CMPG

Build:

The naturally occurring MAPK pathway and quorum sensing mechanism in yeast respond to the activation of α-pheromone, involving pprm1 and downstream genes, and play a role in membrane fusion during yeast mating.Based on this principle, we have devised a system that receives copper ion signals and translates them into α-pheromone signals. Through the MAPK pathway, we express the green fluorescent protein (GFP) gene, thus achieving the detection of copper ions. As illustrated in Figures 1-3.

We utilize the pCUP1 and mfα2 elements to convert the copper ion signal into a pheromone signal. By leveraging the MAPK pathway, we amplify this signal, and ultimately convert the pheromone signal into a green fluorescent output through the pprm1 and GFP elements.

Figure 1-3: Recombinant Plasmid pRS415-CMPG

Test:

Construct plasmid pRS415-CMPG, transform it into Escherichia coli, and culture for a period of time. Purify the plasmid and validate it through gel electrophoresis before electroporating it into yeast. PCR and gel electrophoresis were used for validation, and characterization was conducted using flow cytometry. The results are shown in Figures 1-4.

Figure 1-4: Relationship between Copper Ion Concentration and GFP Expression Intensity in the CMPG Circuit

Learn:

From the flow cytometry data, it is evident that as the copper ion concentration increases, the relative expression also rises, reaching a peak value before starting to decline. Our pprm1 promoter has successfully lowered the basal expression level of pCUP1, yet our genetic circuit appears to be insufficiently sensitive to the gradient of copper ion concentrations. In our upcoming modifications, we aim to enhance its sensitivity and further reduce its basal expression to refine the sensor's performance.

Design:

The plasmid pRS415-CMPG shows promise in achieving the detection objective for copper ions. However, there are challenges with high basal expression and insufficient amplification effects. To enhance the sensor's capability for sensitive and precise copper ion detection, we aim to further reduce basal expression and enhance amplification effects. For this purpose, we have devised two optimization strategies:
① Replacement of pprm1 with a More Sensitive Promoter: We propose to replace the pprm1 promoter with a more sensitive pprm1 Pro promoter. This replacement is expected to enhance the sensitivity for recognizing pheromones, resulting in the design of CMPG Pro.
② Utilization of a Positive Feedback Loop: We plan to construct a positive feedback loop using the Ste5ΔN-CTM protein. This loop would be controlled by an inducible pheromone-responsive promoter, pprm1, which would activate the amplification effect of the MAPK pathway. This approach aims to further enhance the sensitivity for recognizing pheromones, resulting in the design of CMPS.

Figure 2.1-1 Design of CMPG Pro

Build:

We inserted fragments of pCUP1 , mfα2, and CYC1 into the HindⅢ enzyme-digested plasmid pRS415-pprm1 Pro-GFP-CYC1, resulting in the construction of plasmid pRS415-CMPG Pro. Building upon the foundation of pRS415-CMPG, plasmid pRS415-CMPG Pro incorporates modifications to convert pprm1 into pprm1 Pro, aiming to reduce basal expression and enhance amplification effects. This is illustrated in Figure 2-2.

Figure 2.1-2: Recombinant Plasmid pRS415-CMPG Pro

Test1:

Construct plasmid pRS415-CMPG Pro, transform it into Escherichia coli, and culture for a period of time. Purify the plasmid and validate it through gel electrophoresis before electroporating it into yeast. PCR and gel electrophoresis were used for validation, and characterization was conducted using flow cytometry. The results are shown in Figure 2-3.

Figure 2.1-3: Relationship between Copper Ion Concentration and CMPG Pro Circuit GFP Expression Intensity

Learn1:

Overall, the induction expression of plasmid pRS415-CMPG Pro has shown improvement, endowing the sensor with higher performance. It further reduces basal expression, but the amplification effect of the signal has seen limited improvement, leading to inadequate sensitivity enhancement. In our forthcoming modifications, we aim to enhance its sensitivity and further reduce its basal expression to refine the sensor's performance.

Design:

Building upon the foundation of CMPG, the objective of CMPS is to enhance the amplification effect of the circuit, thereby expanding the dynamic detection range.

Figure 2.2-1 Design of CMPS

Build:

We inserted fragments of pprm1, Ste5ΔN-CTM, and CYC1 into the NotⅠ enzyme-digested plasmid pRS415-CMPG, resulting in the construction of plasmid pRS415-CMPS. Ste5ΔN-CTM, a modified version of Ste5 with its N-terminal removed and the CTM gene inserted at the C-terminal, is integrated into plasmid pRS415-CMPG. Ste5ΔN-CTM can self-anchor to the membrane, maintaining the MAPK pathway in an open state. This achieves an expanded dynamic detection range and enhanced sensitivity. This is illustrated in Figure 2.2-2.

Figure 2.2-2: Recombinant Plasmid pRS415-CMPS

Test:

Construct plasmid pRS415-CMPS, transform it into Escherichia coli, and culture for a period of time. Purify the plasmid and validate it through gel electrophoresis before electroporating it into yeast. PCR and gel electrophoresis were used for validation, and characterization was conducted using flow cytometry. The results are shown in Figure 3-3.

Figure 2.2-3: Relationship between Copper Ion Concentration and CMPS Circuit GFP Expression Intensity

Learn:

CMPS exhibits higher expression even for lower copper ion concentrations, expanding the dynamic detection range and significantly improving sensitivity. However, there is a slight increase in basal expression.

Design:

Building upon the modifications from CMPG to CMPG Pro and CMPS, each achieved success in different aspects of the sensor. Considering the successful outcomes of both strategies, we envision combining them to create a sensor that possesses both the low basal expression of CMPG Pro and the sensitivity of CMPS.

Figure 3-1 Design of CMPS Pro

Build:

Fragments of pprm1 pro, Ste5ΔN-CTM, and CYC1 were inserted into the NotⅠ enzyme-digested plasmid pRS415-CMPG Pro, resulting in the construction of plasmid pRS415-CMPS Pro. This hybrid plasmid aims to integrate the advantages of both CMPG Pro and CMPS. This is depicted in Figure 4-2.

Figure 3-2: Recombinant Plasmid pRS415-CMPS Pro

Test:

Construct plasmid pRS415-CMPS Pro, transform it into Escherichia coli, and culture for a period of time. Purify the plasmid and validate it through gel electrophoresis before electroporating it into yeast. PCR and gel electrophoresis were used for validation, and characterization was conducted using flow cytometry. The results are shown in Figure 4-3.

Figure 3-3: Relationship between Copper Ion Concentration and CMPS Pro Circuit GFP Expression Intensity

Learn:

The results are less satisfactory in the lower concentration range, but promising in higher concentration conditions. However, our expectations were not fully met, potentially due to the higher transcriptional load driven by the pprm1 pro promoter for Ste5ΔN-CTM under low concentrations. Further research will be conducted to address this limitation.

1. Copper ions at different concentrations induced different gene lines to express GFP:

The engineered yeast strain BY4741-pRS415-CMPG/CMPG Pro/CMPS/CMPS Pro samples were processed and detected by flow cytometry, and then the data were processed by Flow jo, GraphPad Prism software. The results are shown in Fig. 4-1. The fluorescence intensity was positively correlated with the concentration of copper ions in a certain concentration range. And CMPG pro and CMPS have positive feedback amplification relative to CMPG, while CMPS pro amplification is better than the other two in some cases. The components are effective, which lines with the design idea.

Figure 4-1 Relationship between Cu²⁺ concentration and intensity of GFP expression in different gene lines

2. At the same concentration (50 μM), copper ions induced different gene lines to express GFP:

The engineered yeast strain BY4741-pRS415-CMPG/CMPG Pro/CMPS/CMPS Pro was pre-cultured and then the samples were induced with 50μM copper ions, treated and detected by flow cytometry. Then the data were processed by Flow jo, GraphPad Prism software. The results are shown in Figure 4-2.

Firstly, the CMPG was able to output fluorescent signals smoothly under copper ion treatment , indicating that the pathway worked. On the basis of CMPG, the relative inducibility of CMPG Pro and CMPS was enhanced by 6.8% and 8.8% respectively, indicating that the two signal amplification strategies - replacing the promoter with the one with a larger dynamic range and adding a positive feedback module - were both feasible. CMPS Pro showed a 9.0% increase in induction capacity compared to CMPG, indicating that the two strategies can work synergistically to further amplify the signal and thus make the sensor more sensitive.

Figure 4-2 Relationship between 50 μM Cu²⁺ concentration and intensity of GFP expression in different gene lines