iGEM 2023 — Team nanjing-bioxstem

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# Notebook

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* [![](https://static.igem.wiki/teams/4779/wiki/biankuang1.jpg)](#section1)
* [![](https://static.igem.wiki/teams/4779/wiki/biankuang2.jpg)](#section2)
* [![](https://static.igem.wiki/teams/4779/wiki/biankuang3.jpg)](#section3)
* [![](https://static.igem.wiki/teams/4779/wiki/biankuang4.jpg)](#section4)
* [![](https://static.igem.wiki/teams/4779/wiki/biankuang5.jpg)](#section5)
* [![](https://static.igem.wiki/teams/4779/wiki/biankuang1.jpg)](#section6)

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* [Abstract](#section1)
* [Target 1: BCM Production](#section2)
* [Target 2: Spider Silk](#section3)
* [Target 3: Dye Production](#section4)
* [Target 4: Yeast Engineering](#section5)
* [Conclusion](#section6)

# Abstract

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.

![](https://static.igem.wiki/teams/4779/wiki/engineering0.jpg)

Figure 1-1 Concept map of design ideas

# DBTL1：

## 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.

![](https://static.igem.wiki/teams/4779/wiki/engineering1.jpg)

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 p*CUP1*
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.

![](https://static.igem.wiki/teams/4779/wiki/engineering2.jpg)

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.

![](https://static.igem.wiki/teams/4779/wiki/engineering3.jpg)

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 p*CUP1*, 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.

# DBTL2.1:

## 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.

![](https://static.igem.wiki/teams/4779/wiki/engineering10.jpg)

Figure 2.1-1 Design of CMPG Pro

## Build:

We inserted fragments of p*CUP1*
, 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.

![](https://static.igem.wiki/teams/4779/wiki/engineer4.jpg)

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.

![](https://static.igem.wiki/teams/4779/wiki/engineering5.jpg)

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.

# DBTL2.2:

## 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.

![](https://static.igem.wiki/teams/4779/wiki/engineering11.jpg)

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.

![](https://static.igem.wiki/teams/4779/wiki/engineering6.jpg)

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.

![](https://static.igem.wiki/teams/4779/wiki/engineering7.jpg)

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.

# DBTL3:

## 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.

![](https://static.igem.wiki/teams/4779/wiki/engineering12.jpg)

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.

![](https://static.igem.wiki/teams/4779/wiki/engineering8.png)

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.

![](https://static.igem.wiki/teams/4779/wiki/engineering9.jpg)

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.

# Summary:

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.

![](https://static.igem.wiki/teams/4779/wiki/result13.jpg)

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.

![](https://static.igem.wiki/teams/4779/wiki/result14.jpg)

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

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{% extends "layout.html" %}
{% block title %}engineering{% endblock %}
{% block lead %}This page must show the attribution form of your project. This includes the work done by each of the student members on your team and any work that was done by people outside of your team, including the host labs, advisors, instructors, and individuals not on the team roster. This requirement is not about literature references - these can and should be displayed throughout your wiki.{% endblock %}
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# Silver Medal Experiment:

## I. Design and construction of expression plasmids pRS415-CMPG/CMPG Pro, pRS415-CMPS/CMPS Pro

1. p*CUP1*
, mfα2, CYC1, pprm1, Ste5ΔN-CTM, pprm1 pro fragments were synthesized by the company.

2. Hind III digested plasmid pRS415- CMPG/CMPG Pro, Not Ⅰ digested plasmid pRS415-CMPS/CMPS Pro

3. Validated by agarose gel electrophoresis

4. Homologous recombination of fragments and vectors into E. coli DH5α and transfection of fragments into E. coli DH5α

5. Chemical transfer of recombinant products into E. coli DH5α

## II. Construction of recombinant strains BY4741-pRS415-CMPG/CMPG Pro, pRS415-CMPS/CMPS Pro

Prepare receptor cells of Saccharomyces cerevisiae BY4741, and introduce the recombinant plasmid into the yeast cells by electrotransfer. Gradient diluted, and coated on SC(-Leu) plates.

## III. Validation of recombinant plasmids pRS415-CMPG/CMPG Pro, pRS415-CMPS/CMPS Pro

Single colonies were picked from the chemotaxis plates of E. coli for culturing access to test tubes containing 5mL LB+5μL of Amp medium, incubated in a shaker for 12-16h. Then the plasmids were extracted to be used as templates for plasmid PCR. After verification by agar gel electrophoresis, the correct samples were sent for sequencing.

## IV. Validation of recombinant strains BY4741-pRS415-CMPG/CMPG Pro, pRS415-CMPS/CMPS

Pro A number of single colonies on yeast electrotransfer plates were selected for transpointing, followed by colony PCR, with primers using M13, colony PCR, and finally validation by agarose gel electrophoresis.

## V. Copper ion induced GFP expression and quantitative analysis

1. Strain pre-culture
In an ultra-clean table, take the bacterial solution, scribe lines on a SC(-Leu) plate and label it. The delineated plates were incubated in a constant temperature incubator at 30°C for 1-2 days for strain recovery. Pick single colonies of recombinant yeast strains BY4741-pRS415-CMPG/CMPG Pro, BY4741-pRS415-CMPS/CMPS Pro on the plates and inoculate into 5 mL of SC(-Leu) liquid medium and label it. Incubate the inoculated liquid medium in a constant temperature shaker for about 18h.

2. Copper ion induced expression of GFP and flow cytometry analysis
Determine the OD600 value of the bacterial solution by UV spectrophotometer, and add a certain amount of bacterial solution into 2mL SC(-Leu) liquid medium so that the OD600 value become 0.1. Then dilute 25mM CuSO4
mother liquor according to the ten-fold gradient and add different concentrations of copper ions into the 2mL bacterial solution with the OD600 value of 0.1. The inoculated test tubes were put into a constant temperature shaker for 24h, and the bacterial solution after 24h of copper ion induction was ready for sample processing and detection by flow cytometry.

3. Time expression spectrum of sensor response to copper ions
Use UV spectrophotometer to measure the OD600 value of the pre-cultured bacterial solution, add 100mL SC(-Leu) medium to the sterilized 250mL conical flask, and the bacterial solution was added so that the starting OD600 value of the final culture fluid system was 0.1. Then a certain amount of 25mM CuSO4
was added to make the final concentration of copper ions in the culture solution was 10 μM. The conical flask was placed into a constant-temperature shaking bed to cultivate the bacterial solution. After that, samples were taken every 2h, OD600 was measured by UV spectrophotometer and GFP expression was measured by enzyme marker until 24h. Sample processing was performed and the sample was detected using flow cytometry.

4. Specificity of the copper ion sensor
Prepare ZnSO4
, MgSO4
, MnSO4
solution of25 mM respectively, and determine the OD600 value of the pre-culture bacterial solution by UV spectrophotometer. Add a certain amount of bacterial solution to the 2mL SC(-Leu) liquid medium, so that the OD600 value of the final culture system was 0.1. Then dilute CuSO4
, ZnSO4
, MgSO4
, MnSO4
solution of 25 mM according to the Ten-fold gradient dilution, add a certain amount of CuSO4
, ZnSO4
, MgSO4
, MnSO4
solution to the 2mL bacterial solution with OD600 value of 0.1 , so that the metal ions concentration of the final culture system was 10 μM. The inoculated tubes were placed into a constant temperature shaker incubation for 24h to process the samples, and then subjected to flow cytometry.

# Gold Medal Experiment:

## I. Machine learning-assisted assembly of inducible and constitutive UAS elements

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 PREs. We defined pprm1 from -280bp to -101bp upstream of the gene start codon as UASprm1, and UAScit at 275nt, as the upstream activation sequence of citrate synthase-encoding gene cit1, which is one of the transcriptional enhancers that have been identified in Saccharomyces cerevisiae. Place these two UAS elements immediately upstream of the original pprm1 to form a new promoter, UASprm1-prm1 and UAScit-prm1 were constructed to obtain high-intensity promoter variants.

## II. Design and construction of expression plasmid pRS415-UASprm1-prm1-GFP-CYC1/pRS415-UAScit-prm1-GFP-CYC1

1.UASprm1-prm1/UAScit-prm1 promoter fragment was synthesized through the company.

2. Plasmid pRS415-GFP-CYC1 was digested by BamHⅠ.

3. Verified by agarose gel electrophoresis

4. Homologous recombination of fragments and vectors.

5. Transfect the recombinant product into E. coli DH5α.

## III. Validation of recombinant plasmid pRS415-UASprm1-prm1-GFP-CYC1/pRS415-UAScit-prm1-GFP-CYC1

Single colonies were picked from the chemotaxis plates of E. coli for culturing access to test tubes containing 5mL LB (Amp) medium and incubated in a shaker for 12-16h. Then the plasmids were extracted and used as templates for plasmid PCR. After verification by agar gel electrophoresis, the correct samples were sent for sequencing.

## IV. Validation of recombinant strain pRS415-UASprm1-prm1-GFP-CYC1/pRS415-UAScit-prm1-GFP-CYC1

Dilute the yeast and prepare receptor cells of Saccharomyces cerevisiae BY4741. Plasmids were electro-transferred into yeast cells. After gradient diluted, the cells were coated on SC(-Leu) plates. A number of single colonies on the yeast electro-transfer plates were selected for trans-spotting, followed by colony PCR, and finally verified by agarose gel electrophoresis. The samples were sent for testing and the correct strains were preserved.

## V. Fluorescence assay of recombinant strain

pRS415-UASprm1-prm1-GFP-CYC1/pRS415-UAScit-prm1-GFP-CYC1
A single colony was picked and connected to SC (-leu) liquid medium, cultured for 20 h. Then dilute it to OD600 of 0.1, cultured at 30℃ for 3 h. Add 5µM pheromone, induced for 1 h, and washed three times by 1×PBS. Detect by flow cytometry.

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{% block title %}results{% endblock %}
{% block lead %}This page must show the attribution form of your project. This includes the work done by each of the student members on your team and any work that was done by people outside of your team, including the host labs, advisors, instructors, and individuals not on the team roster. This requirement is not about literature references - these can and should be displayed throughout your wiki.{% endblock %}
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# Silver medal Results:

## 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.

![](https://static.igem.wiki/teams/4779/wiki/results1.jpg)

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.

![](https://static.igem.wiki/teams/4779/wiki/results2.jpg)

Figure 2-1 Recombinant plasmid pRS415-CMPG

![](https://static.igem.wiki/teams/4779/wiki/results3.jpg)

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.

![](https://static.igem.wiki/teams/4779/wiki/result4.jpg)

Figure 2-3 Recombinant plasmid pRS415-CMPG Pro

![](https://static.igem.wiki/teams/4779/wiki/result5.jpg)

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.

![](https://static.igem.wiki/teams/4779/wiki/result6.jpg)

Figure 2-5 Recombinant plasmid pRS415-CMPS

![](https://static.igem.wiki/teams/4779/wiki/result7.jpg)

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.

![](https://static.igem.wiki/teams/4779/wiki/result8.jpg)

Figure 2-7 Recombinant plasmid pRS415-CMPS Pro

![](https://static.igem.wiki/teams/4779/wiki/result9.jpg)

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.

![](https://static.igem.wiki/teams/4779/wiki/result10.jpg)

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.

![](https://static.igem.wiki/teams/4779/wiki/result11.jpg)

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

![](https://static.igem.wiki/teams/4779/wiki/result12.jpg)

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.

![](https://static.igem.wiki/teams/4779/wiki/result13.jpg)

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.

![](https://static.igem.wiki/teams/4779/wiki/result14.jpg)

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：

![](https://static.igem.wiki/teams/4779/wiki/result15.jpg)

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

CMPG Pro:

![](https://static.igem.wiki/teams/4779/wiki/result16.jpg)

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

CMPS:

![](https://static.igem.wiki/teams/4779/wiki/result17.jpg)

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

CMPS Pro:

![](https://static.igem.wiki/teams/4779/wiki/result18.jpg)

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.

![](https://static.igem.wiki/teams/4779/wiki/result19.jpg)

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.

# Gold medal Results:

## 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.

![](https://static.igem.wiki/teams/4779/wiki/result2-1.jpg)

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)

![](https://static.igem.wiki/teams/4779/wiki/result2-2.jpg)

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.

![](https://static.igem.wiki/teams/4779/wiki/result2-3.jpg)

Figure 3-1 Recombinant plasmid UASprm1-prm1

![](https://static.igem.wiki/teams/4779/wiki/result2-4.jpg)

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.

![](https://static.igem.wiki/teams/4779/wiki/result2-5.jpg)

Figure 3-3 Recombinant plasmid UAScit-prm1

![](https://static.igem.wiki/teams/4779/wiki/result2-6.jpg)

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.

![](https://static.igem.wiki/teams/4779/wiki/result2-7.jpg)

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.

![](https://static.igem.wiki/teams/4779/wiki/resukt2-8.jpg)

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.

![](https://static.igem.wiki/teams/4779/wiki/result2-9.jpg)

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

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