Overview

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Our goal is to design a novel kill switch based on the principles of epigenetic modifications to mitigate the potential environmental risks posed by engineered microorganism. To achieve this vision, we have designed and constructed toxic gene plasmids and epigenetic tool plasmids. These different modules were assembled and tested individually in both S.cerevisiae and E.coli. We assessed their functionality by conducting substance tests using test paper. Based on these efforts, we created a "wind-up cell" that can suppress the function of the toxic gene under specific conditions. However, when the cells are removed from the particular environment, the kill switch is activated and the toxic gene exerts its function, leading to cell death.

This innovative strategy enables genetically engineered microorganisms to only grow in specific environments and prevents their long-term survival in natural surroundings. Importantly, it eliminates the need for additional inducers to induce cell death, making it a highly effective strategy for mitigating the environmental risks associated with genetically engineered microorganisms. Moreover, by exploring different combinations of modifications and toxic genes, it is possible to impart the kill switch with new specific functionalities, thereby offering a wide range of possibilities.

Figure 1. The overview of our design

Epigenetic modification

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Epigenetic modifications entail alterations in the phenotype of a particular gene or DNA region without any changes to the DNA sequence. These modifications typically encompass DNA methylation, histone modifications, non-coding RNAs and so on. The addition of methyl groups to the DNA, mostly at CpG sites, convert cytosine to 5-methylcytosine. When methylated cytosines are present in CpG sites in the promoter and enhancer regions of genes, the genes are often repressed. Histone modification refers to the process of adding or removing chemical modifications on histone proteins through enzymatic reactions. These chemical modifications can influence the spatial conformation of histones, subsequently regulating the accessibility of DNA and post-transcriptional processing^[1].

Epigenetics has been defined and today is generally accepted as “the study of changes in gene function that are mitotically and/or meiotically heritable and that do not entail a change in DNA sequence.^[2]” Therefore, our project utilizes epigenetic modifications as a tool to design a novel kill switch. When the kill switch is activated, the epigenetic modifications suppress the expression of toxic genes, allowing the cells to function normally. During cell subculturing, the epigenetic modifications gradually disappear, leading to the expression of the toxic genes. Based on the hereditary of epigenetic modifications, we speculate that this new mechanism may confer a delayed cell death effect.

DNA methylation

Histone methylation

Histone methylation

Figure 2. The principle of three common epigenetic modifications

Our kill switch in S.cerevisiae

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In the yeast system, we chose two toxic genes, named MazF, and EcoRⅠ, and designed a targeted epigenetic modification tool based on dCas9(BBa_K4703031). Our aim is to inhibit the expression of toxic genes specifically using the tool plasmid. After removing the selection conditions for the tool plasmid, during the plasmid curing process, the modification gradually dissipates, allowing the expression of toxic genes and leading to cell death.

module1 toxic gene

Based on the research, we selected MazF and EcoRⅠ as our choices. MazF functions as a endoribonuclease to interfere with the function of cellular mRNAs^[3]. EcoRI is a restriction enzyme that cleaves DNA double helices at specific sites^[4]. And we place them under the control of a constitutive promoter(BBa_K4703028 and BBa_K4703029) to ensure the inevitable death of the cell.

Figure 3. The principle of the toxic genes. (a) MazF (b) EcoRI

module2 repression tool plasmid

Based on the discovered gene silencing phenomenon in natural organisms, we hope to design targeted tools for specific silencing of toxic genes. Through literature reviews, we have learned that the formation of heterochromatin can cause gene silencing, and extensive research indicates that DNA modifications and histone acetylation levels are associated with heterochromatin formation ^[5]. Therefore, we chose to fuse a histone deacetylase with dCas9, hoping to utilize the specific gene-silencing properties of the fusion protein to construct our kill switch.

a.Tool plasmids constructed based on fusion proteins.

We discovered that histone deacetylases (HDACs) are a class of enzymes that play a significant role in the structural modifications of chromosomes and regulation of gene expression. HDACs promote histone deacetylation, leading to tight binding with negatively charged DNA, compaction of chromatin, and inhibition of gene transcription.

To specifically target the constructed toxic genes, we have chosen the CRISPR system. So dCas9 and gRNA are also important components of our tool plasmids.

Based on this, we aim to fuse dCas9 with the histone deacetylase(BBa_K4703031) to attempt the transcriptional repression of toxic genes by specifically targeting the promoters using gRNA. Simultaneously, we seek to test the suppressive effect of this tool by targeting the promoter of a fluorescent protein.

Figure 4. The schematics of the fusion proteins and gene circuits

b.Tool plasmids constructed based on recruiting epigenetic modification proteins through specific sequences

Using the effect of epigenetic modifications, the following method is based on two specific sequences, HML-I(BBa_K4703006) and HML-E(BBa_K4703007) silencers. It has been found that HML can recruit other silencing factors, foremost the repressive Sir2/Sir3/Sir4 complex, to establish heterochromatin-like structures at the HML loci^[6].

Inspired by this study, we have incorporated these specific sequences on both ends of the toxic gene(we assemble two composite part BBa_K4703025 and BBa_K4703026 using different toxic genes). And we aim to inhibit the expression of the toxic gene using this strategy.

Figure 5. The principle of the specific sequences

Our kill switch in E.coli

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In E.coli, we have also chosen toxic genes and tool plasmids suitable for this bacterium, aiming to construct a kill switch to specific inhibit the expression of toxic gene using the tool plasmid.

module1 toxic gene

We have selected the ccdB gene, which encodes a protein called ccdB, as the toxic gene(BBa_K4244056). The ccdB gene is a double-stranded DNA endonuclease toxin that exerts its toxic effect by inhibiting DNA gyrase, an enzyme crucial for DNA replication and transcription. The ccdB protein has the ability to bind and interfere with DNA gyrase, disrupting normal DNA synthesis and transcription, thereby inhibiting bacterial growth^[7].

module2 repression tool plasmid

Through literature research, we found that Dam methylase can exert certain inhibitory effects on DNA methylation modification of genes in E.coli. Therefore, we hope to use Dam as a epigenetic modification tool for E.coli(BBa_K2142004).

Dam is a methyltransferase that typically exists in Gram negative bacteria such as E.coli. It is widely involved in bacterial DNA modification and genetic regulation processes. Dam methyltransferase is responsible for adding a methyl group to adenine in a specific DNA sequence (GATC) and participating in the methylation modification of bacterial DNA.

We found a dam recognition site in the -35 region and -10 region of the mioC promoter, respectively. Through literature review, it has been found that some DnaA proteins bind to the 9 bp sequence (5'-TTTCCACA-3') of mioC, leading to inhibition of mioC transcription^[8]. We selected the mioC promoter to attempt to suppress the gene.

We choose Dam as a tool to inhibit downstream gene expression, connecting different toxic genes downstream of two specific promoters to form our kill switch.

Correspondingly, CRISPRi is a commonly used tool for inhibiting gene expression.We hope to use CRISPRi to enhance its inhibitory effect on downstream genes. We tested the inhibitory effect of the CRISPRi system on downstream genes and designed two strategies to combine CRISPRi with Dam methyltransferase. In one strategy, Dam and the CRISPRi system were separately introduced into E.coli to exert their individual effects. In another strategy, we tested the effectiveness by fusing Dam methyltransferase with dCas9 to construct a fusion protein(BBa_K4703002) as a tool.

Figure 6. The principle of Dam methyltransferase

Our whole-cell detection system

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In order to test the application of the wind-up cell, we applied E.coli cells containing the kill switch system to a whole-cell test paper. When the test paper was initiated, the kill switch would be immediately activated, triggering a countdown to cell death. We designed a corresponding genetic pathway that allows the cells to express a fluorescent protein when exposed to the target substance during the countdown, thereby indicating the presence of the target substance in the environment.

We planned to use two detection plasmids consisting of different inducible promoters and fluorescent protein genes(BBa_K4703023 and BBa_K4703030), enabling E.coli cells to display the corresponding fluorescence when they encounter the target substance. Strains containing both the kill switch system and the detection plasmid were immobilized on the paper using a cross-linking reaction with sodium alginate and calcium chloride solution, creating a detection zone. Inducer solutions, diluted in LB liquid medium, were added to the detection zone at 0h, 4h, 8h, 12h, and 24h, and the differences in brightness were recorded over time. We aim to further determine the detection threshold of this test paper through quantitative experiments and calibrate the fluorescence intensity at different concentrations using known standards to achieve semi-quantitative detection.

The detection and subsequent validation of substances such as IPTG and tetracycline provide possibilities for applying the kill switch system we constructed to other biosensors. In the future, our project can also be combined with various applications of engineered bacteria to reduce the risks associated with their use, ensuring safer and more effective implementation in applications such as gut microbiota therapy and marine oil degradation. This approach allows engineered bacteria to be promptly terminated after fulfilling their role, thereby avoiding ecological risks associated with their potential spread in the environment.

Figure 7. The schematic of the whole-cell test paper