iGEM 2023 — Team tianjin

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April

[May](#left2)

[June](#left3)

[July](#left4)

[August](#left5)

[September](#left6)

[October](#left7)

April
:   **HP**
    During the campus open day, known as the Magnolia Festival, we showcased the charm of synthetic biology to students, teachers, and the public through a variety of creative formats, making the promotional activities engaging and entertaining.

May
:   **Wet lab:**
    The experiments preliminarily validated that *S.cerevisiae* toxic genes MazF can be used.

    **HP**
    Created the initial draft of the Engineered Microorganism: Under the Sword of Damocles card game.
    Participated in the World Intelligence Conference (WIC) and introduced synthetic biology at our booth. Our theme covered the design and synthesis of artificial genomes, DNA storage, as well as the intelligent, automated, and flexible artificial cell factories.

June
:   **Wet lab**
    *S.cerevisiae*：
    pR\_DK02、pR-DK03plasmid and control plasmid were separately transformed into BY4742 The experimental group showed minimal growth, while the control group grew normally, confirming that toxic genes can kill *S.cerevisiae*.

    Test paper：
    The experimental plan was initially determined.

    **HP**
    Improved and refined the card game rules.
    Commenced a 14-day volunteering program, teaching students in rural areas about synthetic biology.

July
:   **Wet lab:**
    Test paper:
    Initial experiments attempted to replicate test papers according to literature. *E.coli* strains induced by IPTG to produce RFP were successfully immobilized on the test paper, but the desired luminescent effect was not achieved. Attempts to add LB medium to the test paper and take moisture-preserving measures successfully produced visible fluorescence after induction.

    Kill Switch:
    Constructed fusion proteins with dCas and Sir2.
    Attempted to suppress the GFP gene using the constructed fusion protein and observed fluorescence intensity under a fluorescence microscope, confirming its inhibitory effect.
    The plasmid containing the constructed fusion protein was co-transformed with pR-DK03 into BY4742. Compared to the control group, the strain grown showed a significant reduction, proving the effectiveness of EcoRI in killing the strain.
    Conducted research on *E.coli* epigenetic modifications and preliminarily selected methylation as a method to suppress toxic genes.

    **HP：**
    Had our first dialogue with Tianjin High School, introducing the iGEM competition process, touring their laboratory, and listing the necessary equipment for experiments.
    Completed the final version of the Engineered Microorganism: Under the Sword of Damocles card game and put it up for sale on Taobao.
    During a high school summer camp, we taught high school students how to use microbial fluorescent proteins (RFP, GFP) for painting and provided them with hands-on laboratory experience.

    **Dry lab：**
    Through reviewing several research papers, we identified the models to establish and comprehensively considered the relevant factors associated with the models. Eventually, the toxic gene expression model was determined. Based on further in-depth research, we preliminarily constructed multiple dynamic models employing multivariate ordinary differential equations to solve the concentrations of toxic gene transcripts, the levels of epigenetic modifiers, the proportions of modified promoters, the cell death rate, and the expression levels of toxic genes. Literature was investigated to construct the first Cell Death Simulation Model.

August
:   **Wet lab:**
    *S.cerevisiae*:
    Construct pR\_DK04 and pR\_DK05 plasmids,respectively, and transform them into the BY4742. After verification, it was found that the transformed cells grew properly, indicating the suppression of the toxic genes.

    *E. coli*:
    Constructed pR\_DK06 and pR\_DK07 plasmids, transformed into *E. coli*, but sequencing yielded no results.
    Re-extracted pR\_DK06 and pR\_DK07plasmids, and sequencing was successful.
    Transformed these plasmids into MG1655 *E. coli* containing the endogenous Dam methyltransferase gene and JM110 *E. coli* lacking it. Observations showed that the pR\_DK07 suppression effect was contrary to the expected target, so this promoter was eliminated.
    Fluorescence intensity of pR\_DK06 plasmids in both strains was measured using an enzyme-linked immunosorbent assay, preliminarily confirming the methylation suppression effect.
    Attempted to construct pR\_DK09 and pR\_DK14 plasmids.

    Test paper:
    Tested the effect of different inducer concentrations on test paper brightness.

    **HP:**
    We co-hosted an online debate with the BIT-China team from Beijing Institute of Technology (BIT). The debate centered around "The Promotion/Hindrance of Scientific Research by the Formulation of Biosafety and Bioethics Regulations."
    Had our second dialogue with Tianjin High School, where we taught high school students synthetic biology experiments and played card games together.

    **Dry Lab：**
    We have attempted to construct and solve equations for the expression levels of toxic genes and equations related to cell growth and death. The epigenetic modification model and promoter modification equation have been successfully established, laying the foundation for the preliminary form of the final model. In designing the promoter modification equation, we made efforts to consider promoter activity, and we have initially determined the corresponding differential equation forms. The steps for making a Cell Death Simulation Model were improved based on the wet lab experiment test paper.

September
:   **Wet lab:**
    *E. coli*:
    Sequencing results for pK\_DK09 plasmid were correct.
    The toxic gene ccdB segment was synthesized by a company.
    The lab already had pR\_DK12 plasmids transformed into *E. coli*.
    Testing different sgRNAs for the suppression of toxic genes.
    Constructing fusion proteins with dCas and Dam methyltransferase.
    Constructed pR\_DK10 plasmids.

    Test Paper:
    Tested luminescent effects of strains containing pR\_DK12 plasmids immobilized on test paper.
    Tested the influence of different inducer concentrations on fluorescence intensity.
    Tested luminescent effects of strains containing pR\_DK14 plasmids immobilized on test paper but abandoned further testing due to the absence of fluorescence.

    **HP：**
    Completed a promotional comic for community science outreach activities to raise awareness about potential biosafety issues in daily life.
    Produced and published a comic about the history of synthetic biology for science outreach, which was released on our WeChat account.

    **Dry lab：**
    We have successfully designed and established the equation for toxic gene expression. Using computer modeling tools, we integrated the formulas into a application (App) interface, enabling the drawing of curves by dragging control values. Two model graphs were created: one depicting the interaction model between methyltransferase concentration (Met) and unmethylated modified promoter concentration (Pro) over time, and another describing the interaction model between protein content of toxic gene expression and cell count over time. The Cell Death Simulation Model has yielded the final results. Additionally, concise documentation for the experimental procedures has been composed in the Wiki section.

October
:   **Wet lab：**
    Fluorescence verification of CRISPRi suppression effect.

    **Dry lab：**
    The Wiki section related to the dry lab experiments and the software section has been completed, with content optimization carried out for both sections.

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Overview

[Kill Switch](#left2)
+
- [> *S.cerevisiae* Cycle 1](#left3)
- [> *S.cerevisiae* Cycle 2](#left4)
- [> *S.cerevisiae* Cycle 3](#left5)
- [> *E.coli* Cycle 1](#left6)
- [> *E.coli* Cycle 2](#left7)
- [> *E.coli* Cycle 3](#left8)

[Whole-cell test paper](#left9)

[Model](#left10)
+
- [> Toxic gene expression model Cycle 1](#left11)
- [> Toxic gene expression model Cycle 2](#left12)
- [> Toxic gene expression model Cycle 3](#left13)
- [> Cell Death Simulation Model Cycle 1](#left14)

# Overview

---

We aim to independently construct epigenetically regulated kill switches in *S.cerevisiae* and *E.coli*. In a given environment, epigenetic modifications suppress the expression of toxic genes. Upon leaving the specified environment, these epigenetic modifications gradually dissipate, leading to the expression of toxic genes and subsequent cell death. We will employ epigenetic modifications and CRISPR technology to engineer these kill switches in both *Saccharomyces cerevisiae* and *E.coli*. Furthermore, we intend to develop a detection circuit within the wind-up cell and create test strips, enabling the wind-up cell to function during the delayed death phase. This page will provide an exposition of the design details and the iterative process of our project.

# Kill Switch

---

## *S.cerevisiae* Cycle 1

In *S.cerevisiae*, our initial approach involves the construction of a fusion protein consisting of dCas9 and a deacetylase enzyme as an inhibitory tool. This fusion protein is designed to specifically suppress the expression of toxic genes, with the expectation that when the organism departs from a specific environment, toxic gene expression will be enabled, ultimately leading to cell death.

**Design**

We aim to design and construct two types of plasmids. One type of plasmid consists of constitutive promoters and toxic genes specific to *Saccharomyces cerevisiae*. We chose two toxic genes, MazF and EcoRⅠ, and constructed two plasmids, pR\_DK02 (containing MazF expression cassette BBa\_K4703028) and pR\_DK03 (containing EcoRⅠ expression cassetteBBa\_K4703029). The other type is a tool plasmid based on epigenetic modifications, which involves the fusion of dCas9 and histone deacetylase (HDAC) to create the tool plasmid pR\_DK01 (containing dCas9 and HDAC fusion protein expression cassette BBa\_K4703031).

By employing gRNA specifically targeting the promoters of toxic genes, the dCas9 carrying histone deacetylase suppresses toxic gene expression through epigenetic modifications.

[More details](https://2023.igem.wiki/tianjin/design)

![](https://static.igem.wiki/teams/4703/wiki/engineering/engineering1.png)

**Figure 1.** Schematic diagram of the gene pathway of dCas9 and sir2 fusion protein.

![](https://static.igem.wiki/teams/4703/wiki/engineering/engineering2.png)

**Figure 2.** Schematic diagram of the gene pathway of MazF/EcoRI.

**Build**

Tool Plasmid: We designed two primers using the computational tool and used PCR to obtain the sir2 deacetylase sequence. Subsequently, we employed Gibson assembly to connect this sequence with a linker and the dCas9 sequence on the pRS42H vector, resulting in the construction of the tool plasmid pR\_DK01.

Toxic Gene Plasmids: We obtained the MazF sequence from the *Escherichia coli* genome using PCR. We also synthesized a codon-optimized EcoRⅠ enzyme from a commercial source. Subsequently, we inserted these two types of toxic genes into the pRS415 vector downstream of a constitutive promoter. This process led to the generation of two distinct toxic gene plasmids, named pR\_DK02 and pR\_DK03.

[More details](https://2023.igem.wiki/tianjin/protocol)

**Test**

We initially transformed the tool plasmid pR\_DK01 into the *Saccharomyces cerevisiae* strain BY4742. Upon successful validation of the transformants, we designed primers using a computational tool and used PCR to split each toxic gene plasmid into two segments. Subsequently, both segments of the plasmids were simultaneously transformed into the previously validated *Saccharomyces cerevisiae* strain. This method was expected to delay the expression of the toxic genes, allowing the tool plasmid ample time for targeted suppression.

The transformants, confirmed to contain the toxic genes, were cultivated in selective liquid SC medium lacking leucine to induce loss of the tool plasmid through selection pressure.

![](https://static.igem.wiki/teams/4703/wiki/engineering/engineering3-1.png)

**Figure 3.** Validation of toxic gene effects.
a. Transferring toxic gene EcoRⅠ into *Saccharomyces cerevisiae* BY4742 resulted in sterile colony growth.
b. Transferring empty vector pRS415 into *Saccharomyces cerevisiae* BY4742 resulted in abundant colony growth.

[More details](https://2023.igem.wiki/tianjin/results)

**Learn**

Based on the results, it is hypothesized that the lethality effect of the toxic genes may be excessively strong, or the suppression effect of the fusion proteins is relatively weak. Subsequent plans include optimizing the tool to achieve a higher level of suppression, aiming to construct a functional kill switch.

## *Saccharomyces cerevisiae* Cycle 2

*(This achievement is distinct from Contribution for Bronze)*

In order to enhance the suppression of toxic genes more effectively, we conducted a literature review and discovered a novel approach. Certain specific sequences have been found to recruit silencing factors. We anticipate achieving successful suppression of the toxic genes through this method.

**Design**

We conducted research and selected HML-I and HML-E silencers, known for their ability to recruit other silencing factors, particularly the repressive Sir2/Sir3/Sir4 complex, in the establishment of heterochromatin-like structures at the HM loci. Our aim is to incorporate these sequences at both ends of the toxic genes to achieve a stronger suppression effect.

![](https://static.igem.wiki/teams/4703/wiki/engineering/engineering5.png)

**Figure 4.** Schematic diagram of the gene pathway of MazF/EcoRI with HML-I and HML-E silencers inserted at both ends.

[More details](https://2023.igem.wiki/tianjin/design)

**Build**

Initially, we designed two primers using a computational tool. Subsequently, we utilized PCR to extract the HML-I (BBa\_K4703006) and HML-E (BBa\_K4703007) sequences from the *Saccharomyces cerevisiae* genome, while adding 20bp homologous arms to the fragments. We employed the Gibson assembly method to insert the HML-I and HML-E silencers into both ends of the toxic genes in plasmids pR\_DK02 and pR\_DK03. This resulted in the construction of plasmids pR\_DK04 (containing BBa\_K4703025) and pR\_DK05 (containing BBa\_K4703026).

[More details](https://2023.igem.wiki/tianjin/protocol)

**Test**

Plasmids pR\_DK04 and pR\_DK05 were separately introduced into the *Saccharomyces cerevisiae* strain BY4742, resulting in the successful generation of transformants. Subsequently, we performed colony PCR and sequencing on multiple transformants, obtaining several strains with correct sequences. This provided evidence that the method can effectively suppress the expression of toxic genes to some extent.

Subsequently, we subjected several of these transformants to colony PCR and sequencing. We obtained multiple strains with correct sequences, thereby validating the successful suppression using this method.

![](https://static.igem.wiki/teams/4703/wiki/engineering/engineering5-1.png)

**Figure 5.** The growth status of strains containing the kill switch.
a. Transferred the pR\_DK03 plasmid into *Saccharomyces cerevisiae* containing pR\_DK01.
b. Transferred the empty vector pRS415 into *Saccharomyces cerevisiae* containing pR\_DK01 as a control.

![](https://static.igem.wiki/teams/4703/wiki/engineering/engineering6-1.png)

**Figure 6.** Colony PCR verification to validate the above-mentioned transformants.
a. Colony PCR verification to validate the pR\_DK05.
b. Colony PCR verification to validate the pR\_DK04.

[More details](https://2023.igem.wiki/tianjin/results)

**Learn**

By incorporating HML-I and HML-E silencers on both sides of the toxic gene, recruiting deacetylation-associated proteins, **we successfully achieved the suppression of toxic gene expression through epigenetic modifications**.

## *Saccharomyces cerevisiae* Cycle 3

Due to the immediate cell death upon leakage and expression of toxic genes, characterizing the suppression efficiency of regulatory elements becomes challenging. To assess the suppression effect of our regulatory elements on downstream genes, we replaced the toxic gene with sfGFP for characterization purposes. Firstly, we tested the suppression effect on downstream fluorescent proteins using the fusion proteins constructed as mentioned above. Subsequently, we plan to employ two promoters, CHO and CYC1, to evaluate the suppression effect on downstream genes induced by different promoters.

![](https://static.igem.wiki/teams/4703/wiki/engineering/engineering7-1.png)

**Figure 7.** Schematic diagram of the gene pathway of sfGFP with CHO promoter (a) or CYC1 promoter (b).

**Build**

We employed *Saccharomyces cerevisiae* homologous recombination to integrate CHO-sfGFP and CYC1-sfGFP into the *Saccharomyces cerevisiae* genome.

**Test**

Subsequently, we performed colony PCR and sequencing on multiple transformants, obtaining several strains with correct sequences. After 24 hours of cultivation, we conducted Microplate Reader to assess gene expression. The results demonstrated a significant suppression effect of our regulatory elements on downstream fluorescent protein genes. Furthermore, it was evident that different promoters had a notable impact on the suppression of downstream genes.

**Learn**

The aforementioned experiments have substantiated the reliability of our epigenetic modification elements. The experimental results have inspired us to explore the impact of different promoters on the suppression of downstream genes. Consequently, we have relayed these findings to the dry lab team, who will further elucidate the underlying reasons for these differences through modeling. The discovery we have made holds significant implications for our subsequent experiments.

## *E.coli* Cycle 1

*(This achievement is distinct from Contribution for Bronze)*

After conducting a thorough investigation, we have identified DNA methylation as a prevalent epigenetic modification mechanism in *Escherichia coli*. By employing methylation on promoters with specific sequences, regulation of downstream gene expression can be achieved.

The Dam methylase can recognize and bind to Dam methylation sites on the mioC promoter and dnaAP2 promoter.

We harnessed the Dam methylase from the *Escherichia coli* genome to investigate the inhibitory effects of methylation on two promoters by conducting suppression tests on fluorescent protein gene.

[More details](https://2023.igem.wiki/tianjin/design)
![](https://static.igem.wiki/teams/4703/wiki/engineering/engineering8-1.png)

**Figure 8.** Schematic diagram of the gene pathway of RFP with mioC promoter (a) or DnaAP2 (b) promoter.

**Build**

We obtained fragments of the mioC promoter (BBa\_K4703004) and dnaAP2 promoter (BBa\_K4703003) through primer annealing using custom-synthesized primers.

Subsequently, we employed Gibson assembly to connect the mioC promoter and dnaAP2 promoter upstream of the RFP fluorescent protein gene, and these constructs were integrated into the pETDuet-1 plasmid, resulting in the generation of pR\_DK06 (containing the mioC-RFP expression cassette BBa\_K4703020) and pR\_DK07 (containing the dnaAP2-RFP expression cassette BBa\_K4703019) plasmids.

We procured the JM110 strain with a commercial exogenous Dam gene from a supplier, while the MG1655 strain, containing the endogenous Dam methylase, was obtained from our laboratory.

[More details](https://2023.igem.wiki/tianjin/protocol)

**Test**

The pR\_DK06 and pR\_DK07 plasmids were transformed into the JM110 strain lacking the endogenous Dam methyltransferase and the MG1655 strain harboring the endogenous Dam methylase.

After 12 hours of cultivation, fluorescence intensity was measured using a Microplate Reader.

It is evident that the fluorescence intensity of JM110 transformants carrying pR\_DK06 was lower than that of the MG1655 transformants. Conversely, the fluorescence intensity of JM110 transformants containing pR\_DK07 was higher than that of the MG1655 transformants.

![](https://static.igem.wiki/teams/4703/wiki/engineering/engineering9-1.png)

**Figure 9.** The growth status of MG1655 and JM110 strains containing the pR\_DK06 plasmid or the pR\_DK07 plasmid.

![](https://static.igem.wiki/teams/4703/wiki/engineering/engineering10-1.jpg)

**Figure 10.** Fluorescence intensity of JM110 and MG1655 cultured for 12 hours.

[More details](https://2023.igem.wiki/tianjin/results)

**Learn**

Experimental results have indeed demonstrated that the Dam methylase can suppress downstream gene expression of mioC promoter. However, it was unable to achieve suppression of downstream gene expression when acting on the dnaAP2 promoter. Consequently, for subsequent experiments, **we have chosen to focus on further investigations involving the mioC promoter**.

The Dam methylation of the mioC promoter has exhibited a certain degree of inhibitory effect on downstream genes. We aim to enhance the suppression effect through further research and explore methods to assist epigenetic modifications in achieving better repression outcomes.

## *E.coli* Cycle 2

After conducting research, we have identified CRISPRi as a commonly used tool for inhibiting gene expression. Therefore, we intend to utilize CRISPRi to complement epigenetic modifications for enhanced suppression of downstream genes.

**Design**

To avoid excessive plasmid introductions, we plan to combine the dCas9 plasmid and sgRNA scaffold plasmid from the commonly used CRISPRi system into a single plasmid. Transcriptional expression of dCas9 will be controlled using a lactose promoter to achieve control over the kill switch.

Considering the issue of plasmid enrichment, we have selected ccdB (BBa\_K4244056) as the toxic gene because ccdB-tolerant strains can be purchased. To achieve better targeted inhibition, we have designed multiple sgRNAs, each targeting the ccdB promoter and open reading frame, respectively.

![](https://static.igem.wiki/teams/4703/wiki/engineering/engineering11-2.jpeg)

**Figure 11.** Schematic diagram of the gene pathway of dCas9 with trc promoter.

[More details](https://2023.igem.wiki/tianjin/design)

**Build**

We employed Gibson assembly to connect the sgRNA scaffold to the plasmid regulated by the trc promoter (BBa\_K3121004) for dCas9 (BBa\_K4703000). Subsequently, we used the Golden Gate method to replace different sgRNAs, resulting in the generation of various plasmids.
The mioC promoter and ccdB toxic gene were commercially synthesized and assembled onto a vector to obtain the plasmid mioC-ccdB (pR\_DK11).

The mioC promoter and ccdB toxic gene were commercially synthesized and assembled onto a vector to obtain the plasmid mioC-ccdB (pR\_DK11).

To enrich the pR\_DK11 plasmid, we purchased the DB3.1 strain, which is tolerant to the ccdB protein, and performed the plasmid enrichment in DB3.1.

[More details](https://2023.igem.wiki/tianjin/protocol)

**Test**

We validated the toxicity of ccdB using MG1655 and found that MG1655 strains containing the ccdB gene exhibited minimal growth.

The constructed tool plasmids were transformed into MG1655. After selecting the correct transformants, susceptible cultures were prepared under 1000X induction. Then, the pR\_DK06 plasmid was introduced, and the cultures were incubated for 18 hours. Fluorescence intensity was measured using a fluorescence quantitative plate reader.

![](https://static.igem.wiki/teams/4703/wiki/engineering/engineering10-2.jpg)

**Figure 12.** Fluorescence values measured under different conditions after 18 hours of cultivation.

[More details](https://2023.igem.wiki/tianjin/results)

**Learn**

**CRISPRi has indeed demonstrated a pronounced inhibitory effect. In the next step, we aim to combine CRISPRi with enhanced epigenetic modifications to achieve stronger suppression of downstream genes.**

## *E.coli* Cycle 3

We plan to enhance the suppressive effects of epigenetic modifications and combine them with CRISPRi to achieve a combined inhibition of downstream gene expression.

**Design**

We plan to introduce exogenous Dam methylase to co-express with endogenous Dam, reinforcing the suppressive effects of epigenetic modifications on gene expression.

We have designed two strategies combining exogenously introduced Dam with CRISPRi:

Approach 1: Dam and the toxic gene are constructed on the same plasmid, and their expression is controlled by the T7-lac promoter (BBa\_R0187). The same inducer is utilized for simultaneous control of epigenetic modifications and CRISPRi.

Approach 2: A fusion protein of dCas9 and DAM (BBa\_K4703002) is constructed. Guided by sgRNA, this fusion protein targets the specific sequence, achieving the inhibitory effect on the target site.

![](https://static.igem.wiki/teams/4703/wiki/engineering/engineering13-1.png)

**Figure 13.** Schematic diagram of the gene pathway of our Option.

[More details](https://2023.igem.wiki/tianjin/design)

**Build**

Approach 1: The dam gene, obtained from the *Escherichia coli* genome, was assembled onto the mioC-RFP plasmid using Gibson assembly, resulting in the plasmid mioC-RFP-Dam (pR\_DK10).

Approach 2: Through Gibson assembly, Dam was connected downstream of dCas9 to construct the fusion protein plasmid pR\_DK08.

[More details](https://2023.igem.wiki/tianjin/protocol)

**Test**

We transferred the plasmids constructed using Gibson assembly into *Escherichia coli*. We successfully constructed pR\_DK08 and obtained transformants containing the pR\_DK08 plasmid.

[More details](https://2023.igem.wiki/tianjin/results)

**Learn**

We transferred the plasmids constructed using Gibson assembly into *Escherichia coli*. We successfully constructed pR\_DK08 and obtained transformants containing the pR\_DK08 plasmid.

# Whole-cell test paper

---

*(This achievement is distinct from Contribution for Bronze)*

We plan to create a whole-cell test paper based on the wind-up cell containing a kill switch as the core. When the test paper is activated, the kill switch is triggered immediately, initiating a countdown for cell death. The wind-up cell will function during the delayed cell death period, ensuring safer substance detection.

**Design**

We intend to utilize plasmids consisting of IPTG-inducible promoters coupled with the RFP fluorescent protein gene and TET-inducible promoters coupled with the GFP fluorescent protein gene as the detection circuit for our whole-cell assay.

To explore the detection threshold of the whole-cell assay, we plan to perform gradient dilutions of inducers to test the fluorescence intensity at different concentrations.

Furthermore, considering that the usage environment of the assay may not be strictly sterile, in order to better reflect the practical working conditions of the assay, we intend to conduct experiments both in sterile and non-sterile environments.

[More details](https://2023.igem.wiki/tianjin/design)

**Build**

We obtained the lac-RFP (pR\_DK13) plasmid and tet-GFP (pR\_DK12) plasmid from the laboratory. The transformants of the former emit red fluorescence under IPTG induction, while the transformants of the latter emit green fluorescence under doxycycline induction.

![](https://static.igem.wiki/teams/4703/wiki/engineering/engineering14-1.jpg)

**Figure 14.** Schematic diagram of the gene pathway of pR\_DK12 (a) pR\_DK13 (b) and pR\_DK14 (c).

We constructed the tet-RFP (pR\_DK14) plasmid through enzyme digestion and ligation. The transformants of this plasmid emit red fluorescence under doxycycline induction.

Cell immobilization: We fixed the engineered bacteria on the test paper through a crosslinking reaction with a solution of sodium alginate and calcium chloride to form the detection zone.

[More details](https://2023.igem.wiki/tianjin/protocol)

**Test**

We placed the test paper into a petri dish and followed the protocol to immobilize suitable bacterial cells on the test paper. After induction, we recorded the fluorescence under a blue light illuminator at specified time intervals.

![](https://static.igem.wiki/teams/4703/wiki/engineering/engineering15-1.png)

**Figure 15.** Test paper result chart of lac-RFP.

![](https://static.igem.wiki/teams/4703/wiki/engineering/engineering16-1.png)

**Figure 16.** Test paper result chart of tet-RFP.

To meet user’s requirements, we repeated the aforementioned detection procedures in a non-sterile environment. The results indicate that, in comparison to test paper produced under aseptic conditions, there was no significant reduction in the fluorescence intensity of the test paper.

[More details](https://2023.igem.wiki/tianjin/results)

**Learn**

We successfully fabricated whole-cell test paper. Among them, the whole-cell test paper containing pR\_DK13 and pR\_DK12 plasmids exhibited fluorescence upon induction, while those containing pR\_DK14 plasmid did not. Therefore, we chose pR\_DK13 and pR\_DK12 plasmids for subsequent testing.

Our whole-cell test paper displayed fluorescence in a contaminated environment outside the clean bench that was not significantly different from the results obtained within the clean bench. Thus, the whole-cell test paper we produced are suitable for use in both contaminated and sterile environments.

# Model

---

In the course of our project development, our team iteratively refined the dry experimentation models using the design-build-test-learn methodology. Serving as the core of our software and model development, we engaged in iterative processes of algorithms and models. We meticulously compared the computational data generated by our models with the results from wet lab experiments, conducting a thorough comparative analysis to assess the accuracy of our models. This iterative cycle persisted, culminating in the successful creation of two pivotal models: a **toxic gene expression model and a Cell Death Simulation Model**.

To explore our software toolkit, please visit our Gitlab repository.

## Toxic gene expression model Cycle 1

*(This achievement is distinct from Contribution for Bronze)*

The toxic gene expression model emerged as a product of our meticulous alignment between dry and wet experimentation processes.

**Design**

We engaged in in-depth, face-to-face discussions with the wet lab team, thoroughly understanding their experimental procedures. Subsequently, we conducted extensive literature reviews, comprehensively considering various influencing factors relevant to the model. Through this process, we precisely defined the direction and objectives for establishing the toxic gene expression model.

**Build**

Building upon intensive research, we initially formulated multiple dynamic models, employing systems of multivariate ordinary differential equations to solve key parameters such as toxic gene transcription product concentration, epigenetic enzyme concentration, promoter modification ratio, cell death rate, and toxic gene expression levels.

**Test**

During the testing phase, we endeavored to integrate these models using computer modeling tools. Initially, we attempted to describe toxic gene expression levels using the Hill function, generating corresponding visual representations. However, significant disparities were observed between these outcomes and the observations from wet lab experiments.

**Learn**

We recognized the pivotal role of specific model selection in accurately depicting the experimental process. To enhance precision in our descriptions, we discerned the imperative need for the model involving promoter modification by methyltransferase to more closely align with our wet lab operational procedures and the established toxic gene expression model.

This meticulous approach and continuous refinement facilitated the development of our robust toxic gene expression model, bridging the gap between theoretical frameworks and real-world experimental nuances.

## Toxic gene expression model Cycle 2

**Design**

In order to precisely delineate the processes of epigenetic modification, toxic gene expression, and cell growth, we reengineered the models associated with these aspects. This redesign aimed at more accurately predicting toxic gene expression levels and the dynamics of cell growth and death.

**Build**

Using computer modeling tools, we meticulously modeled the new set of equations, generating corresponding code. Subsequently, we developed a application (app) tailored for model manipulation and analysis, ensuring seamless integration and functionality.

**Test**

The newly formulated model code was coupled with specific parameter values to generate graphical representations. Comparative analysis was then conducted, aligning the model outputs with experimental results. Through iterative optimization, the model outputs gradually converged with the experimental data. Additionally, wet lab experiments revealed a correlation between the strength of epigenetic modifications and promoter activity.

**Learn**

To accurately capture the relationship between promoter activity and epigenetic modification effects, we recognized the need to introduce a standardized model linking promoter activity with epigenetic modifications. This step was essential for a comprehensive understanding of their intricate interplay, forming the foundation for further refinement.

## Toxic gene expression model Cycle 3

*(This achievement is distinct from Contribution for Bronze)*

**Design**

The introduction of the parameter alpha, correlated with the inherent characteristics of the tools used, facilitated a more nuanced depiction of the relationship between promoter activity and epigenetic modification effects. This inclusion allowed for a finer-grained analysis of their mutual interactions.

**Build**

During application development, we seamlessly incorporated new code into the existing app, adjusting its controls. This effort resulted in the creation of an updated version of the application, enhancing its capability to meet our evolving requirements. The update primarily focused on the refinement and optimization of model parameters.

**Test**

Comprehensive testing was conducted under varied conditions: maintaining consistent promoter activity while altering epigenetic tools, and vice versa. These tests yielded invaluable experimental data, enabling a profound analysis of the model's performance and behavior in different scenarios

**Learn**

The testing process underscored the significance of optimizing model outputs to achieve a higher degree of accuracy and reliability. This realization serves as a pivotal guiding principle for our subsequent research and improvement endeavors, directing our focus towards continual enhancements and advancements in our modeling approach.

## Cell Death Simulation Model

*(This achievement is distinct from Contribution for Bronze)*

**Design**

To simulate the macroscopic process of our test paper entering the environment to be tested - the death of toxic protein-expressing cells, we established a cell death simulation model.

**Build**

We used Python to write code for the cell death model, including the cell distribution cellular automaton model, inducer diffusion model, and association model.

**Test**

We performed 200 iterations of simulation using the cellular automaton model, obtaining simulated results for cell death on the test paper.

**Learn**

The predictive results of our model reflected some shortcomings in the wet lab experiment design. We proposed some improvement solutions and demonstrated the feasibility of these solutions through model simulations, providing new options for the iteration of wet lab experiments.

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Overview

[Fusion protein of dCas9 and Sir2](#left2)

[Incorporate specific sequences](#left3)

[Test with different promoters](#left4)

[Dam methylation](#left5)

[Combine CRISPRi with Dam methylation](#left6)

[Fusion protein of dCas9 and Dam](#left7)

[Test paper](#left8)

# Overview

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We attempted to construct kill switches in both Saccharomyces cerevisiae and *E. coli* and achieved some success. In Saccharomyces cerevisiae kill switch construction, we utilized an epigenetic modification system to suppress the toxic gene and replaced different promoters, using the sfGFP reporter plasmid to assess the gene suppression efficiency. In *E. coli* kill switch construction, we employed epigenetic modifications and CRISPRi technology to suppress the toxic gene, used the RFP reporter plasmid to assess gene suppression efficiency, and continually reduced gene leaky expression. This page will present experimental details of our construction of kill switches in *Saccharomyces cerevisiae* and *E. coli*.

# Create epigenetic modification tool through the fusion of dCas9 and Sir2

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Using computational tool, we designed primers to obtain dcas9 and sir2 deacetylase enzymes through PCR. These enzymes were subsequently linked together using a linker sequence. Additionally, we designed gRNA sequences targeting GFP to construct a tool plasmid. This tool plasmid was then introduced into the *Saccharomyces cerevisiae* strain BY4742. Subsequently, we observed the fluorescence intensity under a microscope.

![](https://static.igem.wiki/teams/4703/wiki/results/result-gfp.png)

# Enhance epigenetic modification elements by incorporating specific sequence

---

We designed primers using a computational tool and performed PCR to obtain MazF and EcoRⅠ genes with homologous arms from the toxic gene plasmids. Subsequently, we obtained HML-I and HML-E silencer from the *Saccharomyces cerevisiae* genome and successfully constructed plasmids pR\_DK04 and pR\_DK05 by Gibson assembly, combining the toxic genes with HML-I and HML-E silencer, and the vector. These plasmids were then separately introduced into *Saccharomyces cerevisiae* BY4742, resulting in the successful transformation of the strains.

![](https://static.igem.wiki/teams/4703/wiki/results/5.png)

**Figure 5.** Strain growth status containing HML-I and HML-E silencer along with a kill switch. a. Transferring pR\_DK04 into *Saccharomyces cerevisiae* strain BY4742 resulted in a great number of transformants. b. Transferring pR\_DK05 into *Saccharomyces cerevisiae* strain BY4742 resulted in a great number of transformants.

Subsequently, we conducted colony PCR and sequencing on multiple transformants, obtaining several strains with correct sequences. This confirmed the successful suppression of the toxic genes using the proposed method.

![](https://static.igem.wiki/teams/4703/wiki/results/6.png)

**Figure 6.** Colony PCR verification to validate the above-mentioned transformants. a. Colony PCR verification to validate the pR\_DK05. b. Colony PCR verification to validate the pR\_DK04.

# Testing tool components by replacing the promoter

---

We used sfGFP as a reporter element to measure the suppression efficiency and employed two different promoters: the CHO promoter and the CYC1 promoter. We measured their fluorescence values before and after the tool element's functionality. We observed a significant decrease in fluorescence values, indicating that the tool plasmid can effectively suppress gene expression. Furthermore, when using the CYC1 promoter, which is weaker compared to CHO, we observed a more pronounced reduction in fluorescence values. This suggests that switching promoters can enhance the degree of gene suppression by the tool plasmid.

![](https://static.igem.wiki/teams/4703/wiki/results/7.png)

**Figure 7.** Suppression of the sfGFP gene with CHO promoter and CYC1 promoter by the tool components

# Inhibiting gene expression through Dam methylation

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We designed primers using a computational tool and employed synthesized primers from Beijing Tsingke Biotech company for annealing. This allowed us to obtain fragments of the mioC and dnaAP2 promoters. The results are illustrated in the accompanying figure.

![](https://static.igem.wiki/teams/4703/wiki/results/8.jpg)

**Figure 8.** Verification of mioC (a) and dnaAP2 (b)

Using seamless cloning, we linked the mioC and dnaAP2 promoters upstream of the RFP fluorescent protein, resulting in the plasmids pR\_DK06 and pR\_DK07. We conducted colony PCR analysis to confirm the connection of mioC and dnaAP2 to the upstream region of RFP.

![](https://static.igem.wiki/teams/4703/wiki/results/9.png)

**Figure 9.** Growth status of *E. coli* after transformation. a. pR\_DK06 transferred to *E. coli*. b. pR\_DK07 transferred to *E. coli*.

![](https://static.igem.wiki/teams/4703/wiki/results/10.png)

**Figure 10.** Verification of pR\_DK06. a. pR\_DK06 upstream. b. pR\_DK06 downstream

We separately transformed the extracted pR\_DK06 and pR\_DK07 plasmids into *E.coli* strains MG1655 and JM110. The results are depicted in the figure

![](https://static.igem.wiki/teams/4703/wiki/results/11.png)

**Figure 11.** Growth status of plasmid transformed *E. coli*. a. pR\_DK06 transferred to JM110. b. pR\_DK06 transferred to MG1655. c. pR\_DK07 transferred to JM110. d. pR\_DK07 transferred to MG1655.

We transformed the pR\_DK06 and pR\_DK07 plasmids into two different *E.coli* strains, namely JM110, which lacks the endogenous Dam methyltransferase, and MG1655, which possesses the endogenous Dam methyltransferase.

It can be observed that the fluorescence intensity of JM110 transformants with pR\_DK06 was higher than that of MG1655 transformants. However, the fluorescence intensity of JM110 transformants with pR\_DK07 was lower than that of MG1655 transformants.

![](https://static.igem.wiki/teams/4703/wiki/results/12.png)

**Figure 12.** The growth status of MG1655 and JM110 strains containing the pR\_DK06 plasmid or the pR\_DK07 plasmid

Preliminary experiments indicate that the Dam methyltransferase can indeed inhibit downstream gene expression of the mioC promoter but does not exert inhibition on the dnaAP2 promoter. Therefore, mioC was selected for further investigation in subsequent experiments.

To further validate the inhibitory effect of methylation on the mioC promoter, we measured the OD values of strains containing the Dam methyltransferase (MG1655) and strains with Dam methyltransferase knocked out (JM110) after 12 hours of culture using an microplate reader. Additionally, we quantified the fluorescence intensity of the pR\_DK06 plasmid expression. We also calculated the fluorescence intensity per unit bacterial concentration. The results indicated that the fluorescence intensity of pR\_DK06 plasmid expression in MG1655 strains, where Dam methylation was present, was lower compared to the normal expression of pR\_DK06 plasmid in JM110 strains. This preliminary experiment suggests that Dam methylation has an inhibitory effect on the mioC promoter.

![](https://static.igem.wiki/teams/4703/wiki/results/13.png)

**Figure 13.** Unit fluorescence values of JM110 and MG1655 containing pR\_DK06

# Enhancing gene repression by combining CRISPRi with Dam methylation

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We transformed plasmids containing the ccdB gene into *E. coli MG1655*, using MG1655 with the plasmid pR\_DK06 as a blank control. It was observed that the *E. coli* cells transformed with the ccdB gene exhibited minimal to no growth.

![](https://static.igem.wiki/teams/4703/wiki/results/14.png)

**Figure 14.** Verification of the ccdB toxic gene. a. MG1655 with ccdB. b. MG1655 with pR\_DK06.

We employed Gibson assembly to ligate the sgRNA scaffold onto a plasmid regulated by the lacZ promoter that controls dCas9 expression. Subsequently, we validated this construction through primer design.

![](https://static.igem.wiki/teams/4703/wiki/results/15.png)

**Figure 15.** Validation of five different tool plasmids through PCR

After selecting single colonies with the correct band size, we extracted the plasmids and conducted sequencing. This process yielded correctly sequenced tool plasmids. Subsequently, we utilized Golden Gate assembly to interchange different sgRNAs, resulting in the generation of five distinct tool plasmids.

![](https://static.igem.wiki/teams/4703/wiki/results/16.png)

**Figure 16.** MG1655 with pR\_DK15 and pR\_DK06.

The mioC-1 tool plasmid (pR\_DK15) was introduced into MG1655 and selected on a culture medium containing chloramphenicol. After obtaining the correct transformants, they were rendered competent under 1000X induction. Subsequently, the pR\_DK06 plasmid was introduced, and selection was carried out on a culture medium containing ampicillin and chloramphenicol. The correct transformants were then cultured in liquid medium for 18 hours, and the fluorescence intensity was measured using a microplate reader.

![](https://static.igem.wiki/teams/4703/wiki/results/17.png)

**Figure 17.** Fluorescence values measured under different conditions.

# Enhancing gene repression using the fusion protein of dCas9 and Dam

---

We employed the Gibson assembly technique to construct the fusion protein of dCas9 and Dam. Following transformation into *E.coli*, selection was carried out on a culture medium containing chloramphenicol. Colony PCR was performed using designed primers, and single colonies with the correct band size were selected for liquid culture. Plasmid extraction and subsequent sequencing were conducted, resulting in the successful acquisition of the pR\_DK08 plasmid.

![](https://static.igem.wiki/teams/4703/wiki/results/18.png)

**Figure 18.** Verification of dCas9 and Dam fusion protein plasmid

# Design diagnostic test paper

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The fluorescence changes within 24 hours after induction were observed on test strips under the following conditions:

1.IPTG: 1.4 mg/L (1000x), 2.8 mg/L (500x), 14 mg/L (100x), 70 mg/L (20x).

![](https://static.igem.wiki/teams/4703/wiki/results/19.png)

**Figure 19.** Test paper result chart of IPTG-RFP

2.Doxycycline (fourfold serial dilutions): 2 mg/L (1000x), 4 mg/L (500x), 20 mg/L (100x), 100 mg/L (20x).

![](https://static.igem.wiki/teams/4703/wiki/results/20.png)

**Figure 20.** Test paper result chart of tet-RFP

The fluorescence intensity in the detection zone of the test strip reached its peak between 8 to 12 hours after contact with the sample. Subsequently, it gradually increased with time, albeit with a slight decrease in fluorescence intensity. Within a certain range, as the concentration of the inducer increased, the brightness of the fluorescence also increased.

![](https://static.igem.wiki/teams/4703/wiki/results/21.png)

**Figure 21.** The fluorescence effects in cleanroom and non-cleanroom environments.

After 24 hours, the gel was washed, and the wash solution was streaked onto solid culture media containing the corresponding resistance markers and left to incubate overnight. It was observed that the cells remained viable, indicating the possibility of strain leakage. This suggests the need for the application of a kill switch.

![](https://static.igem.wiki/teams/4703/wiki/results/22.png)

**Figure 22.** 24h scribbling of Washing liquid.

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