The Gene Guard device involves three proteins:

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

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

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

Our system will have the holin and endolysin genes together with the Phyto-Route and the Auxin Xpress genes on the plasmid, and the anti-holin gene 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 is 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. This will prevent the spread of the AuxIn plasmid to other cells.

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The key to the success of this device is the correct 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 gene expression is controlled by promoter strength and ribosome binding site (RBS) strength, modelling of the Gene Guard is important for helping us choose an appropriate promoters and RBSs.

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The modelling of the 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 we only consider the steady state.

Holin production in wild type soil bacteria

This part of the 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, the number of plasmids in one bacterium is 50.

2) The literature indicates that cell lysis happens at a holin concentration equal to 1000/cell[1], therefore we used 1000 holin/cell as the threshold concentration for cell lysis in our project.

Based on these assumptions, the kinetics of cell lysis in wild type bacteria was modelled using ordinary differential equations (ODEs) for the holin mRNA and holin protein (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 strength of the promoter and RBS controlling expression of anti-holin on the genome should be higher than the promoter and RBS strength of holin on a single plasmid such that all the holin produced by 300 plasmid copies is 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 is described by Equation 3. We defined the ratio of promoter & RBS strength for anti-holin and holin, respectively as "m", as seen in Equation 4.

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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 (J23100) 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. All the holin proteins 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.

In summary, the modelling of gene guard informed us that the promoter strength and RBS strength for holin and anti-holin can be arbitrarily chosen as long as their ratio (i.e. P_anti-holinK_anti-holin)/(P_holinK_holin) >300), to make it certain, the ratio value 400 was selected for our project.

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[1] Christos G. Savva, Jill S. Dewey, John Deaton, Rebecca L. White, Douglas K. Struck, Andreas Holzenburg and Ry Young. The holin of bacteriophage lambda forms rings with large diameter. Molecular Microbiology 69(4), 784–793. 2008.

[2] http://ctb.pku.edu.cn/main/2011course/Network%20motifs/Sneppen%202010/pdf

[3] A. G. ndling., M. D. Manson, R. Young (2001)‘Holins kill without warning’ 9348–9352 PNAS July 31, 2001 vol. 98 u no. 16

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