I: Design ideas of copper ion biosensor based on Saccharomyces cerevisiae MAPK pathway

We used pCUP1 and mfα2 elements to convert the copper ion signal into pheromone signal, then amplify the signal with the help of MAPK pathway and finally convert the pheromone signal into green fluorescence output through pprm1 and GFP elements.

In order to optimize our gene line to make it more sensitive and efficient, we put forward two different optimization ideas:

① Replace pprm1 with the more sensitive pprm1 Pro promoter to improve the sensitivity of recognizing pheromones and designed CMPG Pro.

② Construct a positive feedback loop using Ste5ΔN-CTM, and used the pheromone-inducible promoter pprm1 to control the expression of Ste5ΔN-CTM protein, which activated the amplification effect of the MAPK pathway and further improved the sensitivity of recognizing pheromones, and designed CMPS.

Finally, we combined these two design ideas to further design the CMPS Pro to improve the overall response sensitivity. Our ultimate goal is to design a sensitive and specific copper ion biosensor for copper ion biomonitoring in Saccharomyces cerevisiae based on the MAPK pathway.

Figure 1-1 Concept map of design ideas

II: Plasmid design and construction

1: Design and construction of expression plasmid pRS415-CMPG:

Insert the pCUP1, mfα2, and CYC1 fragments into the Hind Ⅲ enzyme digested plasmid pRS415-pprm1-GFP-CYC1, and simulate and synthesize the corresponding recombinant plasmids using Snapgene software. As shown in Figure 2-1.

The recombinant plasmid was extracted from several single colonies of E. coli on the chemotaxis plates, and PCR was performed to verify that the primer was M13 and the target fragment was 2542 bp. The results were shown in Figures 2-2. The plasmid with the correct bands was sent to GENEWIZ for sequencing, and the results were correct, and the recombinant plasmid pRS415-CMPG was successfully constructed.

Figure 2-1 Recombinant plasmid pRS415-CMPG

Figure 2-2 PCR electropherogram of plasmid CMPG 1~4: plasmid CMPG; M: 5000Marker Target segment 2524bp, all bands are correctly positioned, samples delivered and sequenced correctly

2: Design and construction of expression plasmid pRS415-CMPG Pro:

Insert the pCUP1, mfα2, and CYC1 fragments into the Hind Ⅲ enzyme digested plasmid pRS415-pprm1 Pro-GFP-CYC1, and simulate and synthesize the corresponding recombinant plasmids using Snapgene software. As shown in Figure 2-3.

The recombinant plasmid was extracted from several single colonies of E. coli on the chemotaxis plates, and PCR was performed to verify that the primer was M13 and the target fragment was 2542 bp. The results were shown in Figures 2-4. The plasmid with the correct bands was sent to GENEWIZ for sequencing, and the results were correct, and the recombinant plasmid pRS415-CMPG Pro was successfully constructed.

Figure 2-3 Recombinant plasmid pRS415-CMPG Pro

Figure 2-4 PCR electropherogram of plasmid CMPG Pro 1~3: plasmid CMPG Pro; M: 5000Marker Target segment 2524bp, 1 and 2 bands are correctly positioned, samples delivered and sequenced correctly

3: Design and construction of expression plasmid pRS415-CMPS:

Insert the pprm1, Ste5ΔN-CTM, and CYC1 fragments into the NotⅠenzyme digested vector pRS415-CMPS, and simulate and synthesize the corresponding recombinant plasmids using Snapgene software. As shown in Figure 2-5.

The recombinant plasmid was extracted from several single colonies of E. coli on the chemotaxis plates, and PCR was performed to verify that the primer was M13 and the target fragment was 5000 bp. The results were shown in Figures 2-6. The plasmid with the correct bands was sent to GENEWIZ for sequencing, and the results were correct, and the recombinant plasmid pRS415-CMPS was successfully constructed.

Figure 2-5 Recombinant plasmid pRS415-CMPS

Figure 2-6 PCR electropherogram of plasmid CMPS 1~5: plasmid CMPS; M: 5000Marker band 3 is correctly positioned, samples delivered and sequenced correctly

4: Design and construction of expression plasmid pRS415-CMPS Pro:

Insert the pprm1, Ste5ΔN-CTM, and CYC1 fragments into the NotⅠenzyme digested vector pRS415-CMPS Pro, and simulate and synthesize the corresponding recombinant plasmids using Snapgene software. As shown in Figure 2-7.

The recombinant plasmid was extracted from several single colonies of E. coli on the chemotaxis plates, and PCR was performed to verify that the primer was M13 and the target fragment was 5000 bp. The results were shown in Figures 2-8. The plasmid with the correct bands was sent to GENEWIZ for sequencing, and the results were correct, and the recombinant plasmid pRS415-CMPS Pro was successfully constructed.

Figure 2-7 Recombinant plasmid pRS415-CMPS Pro

Figure 2-8 PCR electropherogram of plasmid CMPS Pro 1~2: plasmid CMPS Pro; M: 15000Marker All bands are correctly positioned, samples delivered and sequenced correctly

III: Construction of recombinant strains

1: Construction of recombinant strain BY4741-pRS415-CMPG/CMPG Pro:

Several colonies on the yeast turn point plate were picked, and the colony PCR was verified with M13 primer, which was unsuccessful; so the colonies were picked again, connected to 5 ml of SC(-Leu) medium, and cultured at 30°C and 200 rpm for 24 h. The genome was extracted and genomic PCR was performed to verify the target fragment of 2542 bp, and the results were shown in Figures 3-1; PCR reaction solution with correct band positions was sent to GENEWIZ for sequencing, and all the results were correct, so the construction of recombinant strain pRS415-CMPG/CMPG Pro was successfully completed.

Figure 3-1 CMPG, CMPG Pro genome PCR electropherograms M: 5000Marker; 1: CMPG; 2: CMPG Pro

2: Construction and validation of recombinant strain BY4741-pRS415-CMPS/CMPS Pro:

A number of colonies on the yeast turn point plate were picked, and the colony PCR was verified with PSC primers, and the target fragment was 2990bp, and the results were shown in Fig. 3-2 and Fig. 3-3; the PCR reaction solution with the correct position of the bands was sent to GENEWIZ for sequencing, and the results of the sequencing were correct, and the recombinant strain pRS415-CMPG/CMPG Pro was constructed successfully.

Figure 3-2 PCR electropherogram of CMPS colonies. M: 5000Marker; 1~9: CMPS; the position of band 1 is correct, and the sample is sent for sequencing correctly

Figure 3-3 PCR electropherogram of CMPS Pro colonies M: 5000Marker; 1~5: CMPS Pro. All bands were correctly positioned, 2 and 4 were sent for sampling and sequenced correctly.

IV. Copper ion induced GFP expression and quantitative analysis

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

3. Gene line expression of GFP induced by copper ions at different concentrations:

The engineered yeast strain BY4741-pRS415-CMPG/CMPG Pro/CMPS/CMPS Pro samples were processed and grouped to be detected by flow cytometry. Then the data were processed by Flow jo, GraphPad Prism software. The results are shown in Fig. 4-3/4/5/6. The fluorescence intensities were positively related to the concentration of copper ions within a certain concentration range . Above a certain concentration, the fluorescence intensity decreased probably because high concentration of copper ions affects the activity of yeast cells . Therefore, the element is effective and conforms to the design idea.

CMPG：

Figure 4-3 Relationship between copper ion concentration and intensity of GFP expression in CMPG lines

CMPG Pro:

Figure 4-4 Relationship between copper ion concentration and the intensity of GFP expression in the CMPG Pro line

CMPS:

Figure 4-5 Relationship between copper ion concentration and the intensity of GFP expression in the CMPS line

CMPS Pro:

Figure 4-6 Relationship between copper ion concentration and the intensity of GFP expression in the CMPS Pro line

V: Specificity analysis of copper ion sensors

In order to investigate whether the yeast sensor has relative specificity for copper ions, the constructed sensor also detected Mn2+, Mg2+, and Zn2+. The engineered strain BY4741-pRS415-CMPG was induced using these three heavy metal ions, the optimal induction time of 20h and the induction concentration of 10 μM. The results are shown in Figure 5-1.

After significance analysis, the GFP intensity of BY4741-pRS415-CMPG induced by Mn2+, Mg2+, and Zn2+ was not significant when compared with that of the blank control (BY4741-pRS415-CMPG without any metal ion induction), whereas there was a significant difference between the Cu2+-induced BY4741-pRS415-CMPG and the blank control. The above results indicate that the yeast sensor has a relative specificity for copper ion detection.

Figure 5-1 GFP intensity of CMPG treated with different metal ions. Control: CMPG strain without any metal ion induction. The control group was significant with the Cu2+ induced group and not significant with the other metal ion induced group.

I: Design ideas for dynamic range optimization of the pprm1 promoter based on UAS elements:

Under the help of our host professor's research team, we leveraged machine learning techniques to conduct a comprehensive screening of the inducible UAS element, and defined UASprm1. Subsequently, we strategically incorporated the previously recognized transcriptional enhancement element, UAScit, into the study. These two meticulously chosen UAS elements were then positioned directly upstream of the original pprm1, resulting in the creation of novel promoters: UASprm1-prm1 and UAScit-prm1. We engineered gene circuits featuring the GFP (green fluorescent protein) marker, translating promoter activity signals into measurable fluorescence intensity. This optimization was validated using flow cytometry, ensuring the accuracy and effectiveness of our approach.

Figure 1-1 Design.

II: Machine Learning Assisted Screening of Induced UAS Components

Structural analysis of the pprm1 promoter by running the NuPoP algorithm (https://bioconductor.org/packages/NuPoP/) revealed 2 features in the pprm1 promoter: first, it contains multiple nucleosome affinity reduction sequences, which help to enhance promoter activity; The second is that it contains three short consensus PRE. We defined pprm1 from -280bp to -101bp upstream of the gene start codon as UASprm1(Figure 2-1)

Figure 2-1 Structural sketch of induced UAS element UASprm1 III: Design and construction of plasmids

1: Design and construction of expression plasmid pRS415-UASprm1-prm1-GFP-CYC1 Place UASprm1 immediately upstream of the original pprm1 to form a new promoter, called pUASprm1-prm1 promoter The corresponding recombinant plasmids were simulated and synthesized using Snapgene software. As shown in Figure 3-1.

The recombinant plasmid was extracted by picking several single colonies of E. coli on the transfection plate, and PCR was performed to verify that the primer was M13 and the target fragment was 220 bp. The result was shown in Figure 3-2. The plasmid with the correct location of the bands was sent to GENEWIZ for sequencing, and the sequencing results were correct, and the recombinant plasmid UASprm1-prm1 was successfully constructed.

Figure 3-1 Recombinant plasmid UASprm1-prm1

Figure 3-2 M：DL1000 DNA Marker（Vazyme） UASprm1-prm1（220bp）

2: Design and construction of expression plasmid pRS415- UAScit-pprm1-GFP-CYC1 UAScit was placed immediately upstream of the original pprm1 to form a new promoter called pUAScit-prm1 promoter The corresponding recombinant plasmids were simulated and synthesized using Snapgene software. As shown in Figure 3-3.

The recombinant plasmid was extracted from several single colonies of E. coli on the transfer plate, and PCR was performed to verify that the primer was M13 and the target fragment was 315 bp. The results are shown in Figure 3-4. The plasmid with correct band positions was sent to GENEWIZ for sequencing, and the results were correct, so the recombinant plasmid UAScit-prm1 was successfully constructed.

Figure 3-3 Recombinant plasmid UAScit-prm1

Figure 3-4 M：DL1000 DNA Marker（Vazyme） UAScit-prm1（315bp）

IV: Construction of recombinant strains

1: Construction and validation of recombinant strain BY4741- pRS415-UASprm1-prm1-GFP-CYC1

Several colonies of yeast were picked from the transient plate, and the colony PCR was verified with PSC primers, the target fragment was 1880bp. The result was shown in Fig. 4-1; the PCR reaction solution with the correct position of the bands was sent to GENEWIZ for sequencing, and the results were correct, so the recombinant strain BY4741- pRS415-UASprm1-prm1-GFP-CYC1 was successfully constructed.

Figure 4-1

2: Construction and validation of recombinant strain BY4741- pRS415-UAScit-prm1-GFP-CYC1

A number of colonies were picked from the yeast transfection plate, and the colony PCR was verified with PSC primers, the target fragment was 1974bp. The results were shown in Fig. 4-2; the PCR reaction solution with the correct position of the bands was sent to GENEWIZ for sequencing, and the results were correct, and the recombinant strain BY4741- pRS415-UASprm1-prm1-GFP-CYC1 was successfully constructed.

Figure 4-2

V. Fluorescence assay of recombinant strains

The samples of engineered yeast strains BY4741- pRS415-UASprm1-prm1-GFP-CYC1, BY4741- pRS415-UAScit-prm1-GFP-CYC1 were processed and detected by flow cytometry, and then the data were analyzed by Flow jo, GraphPad Prism software, and the results are shown in Figure 5-1. . UASprm1 is a pheromone-inducible element that performs well and stably for expanding the dynamic range of natural promoters. The background expression level of the pprm1 promoter was reduced by 12.3% and the induced expression level was increased by 12.6% in upstream tandem with UASprm1.

For UAScit, the pheromone-inducible synthetic promoter UAScit-prm1, although elevating the background expression level to a certain extent, was able to increase the induced expression level more substantially, with a 13.2% increase in basal activity and a 76.9% increase in induced expression level compared to its corresponding core promoter pprm1.

Figure 5-1 Characterization of the fluorescence intensity of different promoters induced by pheromones