In order to illustrate the dynamics of our idea and to compare the strengths of the promoters required for the transcription of the split ribozymes, a model was built using the MATLAB (MathWorks™) extension "SimBiology". With the help of the highly simplified model, the required strengths and quantities of all the compartments and species involved can be estimated for use in the laboratory, and the process can be optimised and troubleshooting simplified.
It is assumed that an E. coli bacteria is infected by a T4 lambda phage, then recombined. The plasmid is then transcribed, whereby the promoter strengths for the expression of both split ribozyme parts can be varied. The focus was on the promoters BBa_J23101, BBa_J23104, BBa_J23115 and BBa_J23116 of the Anderson Collection. The Berkeley iGEM team's 2006 findings about the relative promoter strengths were applied.
At this point, the model was continued via two pathways, the upper pathway representing existing antibiotic resistance, thus forming a hybrid from the two split ribozyme halves. This instance can be represented in the simplified model by indicating the presence of bacterial RNA. In the bottom pathway, only the split ribozyme connected to the promoter is present because there is no antibiotic resistance in this scenario.
In this case, the bacterial RNA is manually marked as non-existent, resulting in zero amounts of the lower pathway's subsequent products. Thus, resistance can be regarded as a simple switch for the pathway.
The built model shows which qualitative results can be expected with changing ratios (especially promoter strengths). It is difficult to present quantitative results in this context because relative values were used. Additionally, no time-limiting factors such as RNA degradation or the time-dependent change in transcription and translation rates in the course of the cell life cycle were taken into consideration.
Thus, the model is far removed from reality and could be further improved in the future.
However, significant effects of the mentioned parameters on the qualitative outcome is not to be expected, and would only become relevant with increasing complexity.
In this strictly model-based approach, without additional precise laboratory observations, the reaction time was assumed to be t=10s and the units of reacting species as [molecule]. Furthermore, the values of the species (which can be regarded as roughly comparable to the number of species) were normalised to 1, which fits the normalisation of the relative promoter strengths.
It may be demonstrated that the highest concentration of the resistance marker can be expected with the promoter combination BBa_J23101 and BBa_J23104. The lowest expression is expected with the promoter combination BBa_J23115 and BBa_J23116.
