The Gene Guard device involves three proteins:

1) Holin is a protein forms pore complexes in the inner-membrane and these pores allow endolysin to move into periplasmic space.

2) Endolysin is an enzyme that could break down the cell wall and causes cell lysis.

3) Anti-holin binds to holin and prevent it from forming pores.

Our system will have holin and endolysin genes together with auxin genes and chemoreceptor genes on the plasmids, and anti-holin genes on the genome under control of strong promoter. The production of anti-holin will prevent cell lysis of the genetically modified (GM) bacteria by deactivating holin. If the plasmid get horizontal transferred to a different cell without anti-holin on its genome(e.g. any wild type soil bacteria), the expression of holin and endolysin will induce cell lysis and hence prevent it from keeping the plasmid containing auxin gene.

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The key to the success of this device is the right ratio of holin and anti-holin production such that the anti-holin can deactivate all the holin produced in our GM bacteria, and the holin expression level in wild type bacteria is high enough to induce cell lysis. As the gene expression is controlled by promoter strength and ribosome binding site (RBS) strength, therefore modelling of gene guard is important for helping us choosing appropriate promoters and RBS to inhibit and optimize cell behavior in our genetic modified cell and soil bacteria respectively.

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The modelling of gene guard consists two parts, one is the holin production in wild type bacteria and the other part is the deactivation of holin by anti-holin in GM bacteria. Since all the genes in our system are constitutively expressed, thereby we only consider steady state.

Holin production in wild type soil bacteria

This part of modelling focused on deriving the relationships between promoter strength and RBS strength of holin on single plasmid. In order to model this, the following assumptions were made:

1) After horizontal plasmid transfer and duplication, there will be 50 copies of plasmids in one bacterium.

2) The literature indicates that cell lysis happens at holin concentration equals to 1000/cell[1].

Based on above assumptions, the kinetics of cell lysis in wild type bacteria was modeled using ordinary differential equations (ODEs) for the holin mRNA and holin (Equation 1). At steady state (i.e. d[ ] /dt = 0), rearranging Equation 1 with [Holin] = 1000, we obtained an expression for P_holin K_holin as shown in Equation 2.

Holin inhibition in GM bacteria

In GM bacteria the promoter strength and RBS strength of anti-holin on the genome should be stronger than the promoter strength and RBS strength of holin on a single plasmid such that all the holin produced by 300 copies of plasmids should be inhibited, where 300 is the maximum number of plasmids in a single bacterium. In this system, the anti-holin can bind to holin to form a dimer, which is defined as the deactivated holin. The mechanism in the GM bacteria can be described in following ODEs in Equation 3.

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By varying the ratio m in equation 4, we found that the holin concentration in GM bacteria decreased to zero at ratio m ≥ 300 (see Figure 1). Then, we tested m = 400 with the anti-holin promoter we have with strength 13.1 pg RNA/min/μg substrate DNA, the result is displayed in Figure 2.

Fig.1: The holin concentration in GM bacteria vs. ratio m (m = (P_anti-holinK_anti-holin)/(P_holinK_holin)). This graph shows that the intracellular concentration of holin remains at zero if m>300 .

Fig.2: The evolution of different protein species vs. time at m = 400. This graph shows that all the holin in the GM cell are inhibited as the holin concentration remains at 0 all the time, while the holin concentration in wild type bacteria reaches 1000 per cell after 5000 seconds.

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