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iGEM NU Kazakhstan 2024 characterization

The construct AraC-pBAD-MazF includes arabinose induced promoter Arac-pBAD (BBa_K808000) and mazF gene (BBa_K2120008). MazF protein is a toxin for bacterial cells that is applied as controlled suicide circuit. It functions as an mRNA endonuclease which inhibits protein synthesis and leads to cell death. MazF is transcribed under the AraC-pBAD promoter that is induced in the presence of L-Arabinose and inhibited in the presence of glucose [1]. The promoter exhibits low leakage levels and high response to arabinose induction [1]. Our team applied AraC-pBAD-MazF construct in a AraC-AraBAD promoter (BBa_K808000) + RBS (BBa_B0034) + MazF Protein (BBa_K2120008) + Terminator (BBa_B0015) link (Figure 1). In addition to iGEM BIT-China- 2016 team’s AraC-AraBAD + RBS + MazF construct (BBa_K2120006), Terminator sequence was added [2].

Figure 1. AraC-AraBAD promoter + RBS + MazF Protein + Terminator link.

The induction of MazF synthesis was performed by incubating E.coli cells with 0.05%, 0.1%, 0.2%, 0.5%, and 1% arabinose concentrations to activate the AraC-AraBAD promoter. To assess the induction of MazF synthesis, the viability of E.coli cells was measured (Figure 2).

Figure 2. OD600 nm values of transformed E.coli cells incubated with different concentrations of arabinose.

Figure 2 indicates that MazF was expressed even at 0.05% arabinose concentration resulting in the reduced viability of cells (x̄ for the control untreated group = 1.57, x̄ for 0.05% ara group = 1.08). As the concentration of arabinose increased, the expression of MazF increased leading to the gradual decrease of OD600 nm values. The lowest proliferation and highest expression of MazF was observed at 1% arabinose concentration, therefore 1% arabinose concentration is the most optimal for MazF synthesis and bacterial growth inhibition. Additionally, high proliferation of cells was observed in the control group with no arabinose added confirming low leakage of the promoter. Thus it was concluded that the AraC-pBAD promoter is suitable for the synthesis of MazF. The mathematical modeling of MazF protein was performed using an ode15s solver type system in MATLAB Simbiology extension. The MazF protein model includes 11 reactions for MazF synthesis and is modeled on the scale of a single bacterial cell.

Figure 3. The concentration of the MazF mRNA produced against time.

Figure 3 depicts the variation in MazF mRNA concentration over time after the induction with arabinose. The response to arabinose induction and rapid MazF mRNA synthesis is observed in less than 1 hour after the induction. As the time after arabinose induction increases, the concentration of MazF mRNA gradually declines, which can be explained by mRNA endonuclease activity of MazF that also cleaves its own mRNA.

Figure 4. The concentration of the MazF protein against time.

Figure 4 represents the production of the MazF protein over time in response to arabinose induction. The sigmoid- shaped curve of MazF production is observed due to mRNA endonuclease function of MazF mentioned earlier that will cause the cleavage of MazF mRNA and subsequent diminution of MazF protein production.

Figure 5. The viability of the bacterial cells.

Figure 5 illustrates E.coli cell viability after the inhibitory concentration of the MazF protein is reached. The obtained results indicate the continuous decline of cell vitality after accumulation of inhibitory MazF concentration. It can be observed that 50% of the cells die within 4 minutes after required concentration is reached indicating high effectiveness of MazF for bacterial cell growth inhibition. Additionally, iGEM BIT-China- 2016 applied AraC-pBAD-MazF construct in the link with a different RBS coding sequence (BBa_K2120002) [3]. The AraC-AraBAD promoter (BBa_K808000) + RBS (BBa_B0032) + MazF Protein (BBa_K2120008) link was designed. The team also constructed the AraC-AraBAD promoter (BBa_K808000) + RBS (BBa_B0032)+hokD (BBa_K2120003) to compare lethal activity of MazF and HokD toxin proteins. The team incubated transformed E.coli BMTop10 with 10% arabinose and measured OD600 to draw the growth curve [3]. OD600 in E.coli transformed with MazF was significantly lower than for the cells transformed with HokD indicating high lethal activity of MazF protein. Additionally, the control groups for HokD and MazF that were not induced with arabinose showed high proliferation, thereby the AraC-pBAD promoter was not leaky and suitable for synthesis of both toxic proteins. iGEM XMU-China-2020 also applied AraC-pBAD-MazF construct in pBAD/araC promoter (BBa_I0500)-RBS (BBa_B0034) -MazF (BBa_K1096002) -Terminator (BBa_B0015) link (BBa_K3332083) [4]. The team induced E.coli cells with 0.2% arabinose and counted the number of groups of E.coli after incubation and growing of induced cells on the petri plate. The group discovered that the number of E.coli colonies significantly decreased after 5 hours of induction with 0.2% arabinose. Secondly, the non-induced group showed no significant changes in the viability verifying low basal level of MazF expression under AraC-pBAD promoter without inducer.

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