In our project, we have constructed a series of SRRz based heavy metal ion detectors. The gene circuits can be summarized as one circuit shown in the picture.
In the center is a bidirectional promoter Pmer , which can be regulated by MerR homodimer and metal-combined MerR homodimer.
Under normal condition, MerR dimer binds with Pmer and recruits RNA Polymerase for the left part to transcript. While the right part, because of the wrong span between -10 box and -35 box which cause them located at different sides of DNA helix, is silent.
But when metal ion binds to MerR dimer, things come different. MerR dimer changes the configuration of DNA, repressing the transcription of the left part and recruiting RNA Polymerase for transcription of the right part.
On the left side of Pmer, we have put a MerR coding sequence here, which can be expressed constitutively when there is no metal ion. For this part, the model is shown in the flow chart.
Then on the left side is the SRRz lysis gene, which is the responding module in our system. This part is a competitor for Pmer against the former part. The flow chart describes the regulation of MerR dimer and metal and the expression of SRRz without the former part.
Up to now, the metal ion detection and reporter protein expression part is done. The whole process is shown in the following figure. The competition between two directions is obvious.
In this part, we constructed our model by hypothesizing that the permeability of cells can be estimated by degradation rate of compound composing the cellular membrane and cell wall. Besides, there should be a cellular membrane repairing mechanism that is in proportion to the degradation rate. Therefore, we built up the following model to describe the process.
Because cell lysis process usually happens extremely fast, we set a threshold that when the degradation of outer membrane is over 0.5, the cell lyses and intracellular proteins come into the system completely. After the cell lysis, β-galactosidase comes out of the cell, accumulates to a relatively high concentration and produces oNP, a colored substrate which can be easily detected by any kind of photographic equipment.
Eventually, we put all the four parts together, and finished the buildup of the model, which is shown in the flow chart.
Getting such a flow chart isn't a hard work, but parameters fitting can recede one's hairline. Table below is the theoretical fitted value of the parameters used in the model. The match result of the model and the wet lab data is shown in the figure.
As a result, our model successfully predicted the result of wet lab, not only that the protein concentration of SRRz matches the real value, but also that our model realizes a cell lysis process at about 3.6x103, which is approximately an hour, matching the wet lab data. We must greatly thank the Simbiology program of matlab for the help of the modeling work.
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