Overview

To ensure the safety of the kill switch, we selected highly toxic genes during the preliminary research. However, due to uncertainty about whether the suppression element we used controls the expression of the toxic genes, we conducted a series of experiments to assess the feasibility of our delayed death concept for the kill switch.

Furthermore, we aimed to improve the existing whole-cell test paper by utilizing the escape prevention system we designed. After replicating the reported findings in the literature, we successfully designed two additional detection gene circuits.

Kill Switch

In S.cerevisiae

In yeast, we added HML-I and HML-E silencer on either side of the toxic genes MazF, and EcoRⅠ. The HML-I and HML-E silencer recruit the sir2/sir3/sir4 complex, successfully suppressing the constitutive expression of the toxic genes and allowing yeast survival. This demonstrates the feasibility of constructing a kill switch by suppressing toxic gene expression using epigenetic modifications within yeast.

Figure 1. Transferring pR_DK04 and pR_DK05 separately into yeast strain BY4742 resulted in a great number of transformants.

In E.coli

In Escherichia coli, we successfully demonstrated significant inhibition of fluorescent protein expression using epigenetic modification (DAM methylase) and CRISPRi respectively. believe that by enhancing epigenetic modifications and combining them with CRISPRi, we have the potential to suppress the toxic gene ccdB, thus establishing a kill switch.

Figure 2. Fluorescence intensity of Escherichia coli transfected into the mioC-mRFP plasmid - determination using an enzyme-linked immunosorbent assay

Whole-Cell Test Paper

Initially, we simulated the fluorescence intensity in alginate-calcium gel on a 1 square centimeter filter paper and successfully reproduced the findings reported in the literature.

Figure 3. Crown strength in calcium alginate gel containing different concentrations of Escherichia coli

We have successfully developed whole-cell test strips with different detection pathways, and these test strips demonstrate detectable fluorescence in both sterile and bacterial environments. This indicates that our test paper have practical feasibility for substance detection in real-life scenarios.

Professor's suggestion

Our PI, Professor Wu Yi, believes that whole-cell biosensors are a hot research direction in synthetic biology. Applying the biological containment system to whole-cell test papers is a form of combined innovation. The application of epigenetic modifications in synthetic biology may play a significant role in future scenarios. Future work may focus on building multiple epigenetic modification elements and continuously arranging them to optimize the suppression effect of toxic genes. These ideas indicate that our project holds a certain level of feasibility and propose suggestions for the future development of our project.