Team:BHSF ND/Model
2019 IGEM BHSF_NDModel
Digitalizing Bistable Module
The general circuit was implemented in the shape of plasmid construct with the following DNA segment:
TF(The transcription factor that induces P1 activity): Arac for pBAD and XyIs Pc for Pm.
P1: The promoter of interest: pBAD for Arac, and Pm for XyIs Pc.
P2: The promoter starts the translation of sRNA 1, which inhibits the expression of the P 1 and its RBS; it is characterized as PLtetR in the general circuit of the iGEM product.
P3: The promoter starts the translation of the sRNA 2, which inhibits the expression of the P 2 and its RBS ; it is characterized as PA1/04 in the general circuit of the iGEM product.
R1:The repressor inhibits the P3 , which further express the sRNA 2; it is characterized as LacI in the general circuit of the iGEM product.
R2: The promoter starts the translation of the sRNA 2, which inhibits the expression of the P 2 and its RBS ; it is characterized as PA1/04 in the general circuit of the iGEM product .
GOI 1The DNA segment expressed; it is characterized as recombinase ( TP901, Bxb1, PhiC31) in the general circuit of the iGEM product, and its attB and attP locate in the 2nd P 2. Functioning as the irreversible switch in the circuit that re-direction the 2nd P 2.
GOI 2: The DNA segment expressed; it is characterized as Toxin ( ParE, Kid, Colicin E2) in the general circuit of the iGEM product. Functioning as the toxin that decompose the circuit as a conditional suicide.
Inducer is constant through the simulations. Species are measured in molecules. External inhibition of the basal level comes from a sRNA molecule that is being express by a specific promoter (PsRNA). Importantly, the expression of such sRNA was made conditional of the activity of the targeted promoter (Ptarget) to be digitalized. This is a regulated expressed by Ptarget inhibiting PsRNA. Mathematical modelling was done to analyse the dynamical feature of the system at different rate value.
Stimulation Process
The generation and degradation rates ( k ) of inducer is measured:
The binding and unbinding of the inducer to the transcription factor is measured.
The basal transcription rate of Promoter is measured.
The induced transcription rate of Promoter is measured.
The translation and degradation rate of repressor and GOI is measured.
The transcription and degradation rate of sRNA by PsRNA is measured
The repression from sRNA to repressor is measured.
The binding and unbinding, respectively, of repressor to/from PsRNA is measured.
The module can be wrote through chemical reaction, as listed follow
The chemical reaction can achieve the ordinary differentiate equation (ODE), as listed followed
Simulation Result
The results shows that the double negative feedback loop (sRNA represses the regulator; the regulator inhibits the expression of sRNA) allows for digitalisation at specific parameter settings. T he stronger that both regulations are, the more digital that Ptarget response is. If any one of the regulations were weak, final performance would not show the required results. By digital we understand clear on/off states with a sharp transition in between. Moreover, the model suggested that, in the best scenario (i.e. strong repressions), only sRNA or mRNA will be found in She system at any given time, with the exception of a brief time lapse in which both molecules coexist before solving the issue into an stable state. Therefore, this returned a prediction: better to use a strong regulator (repressor)and strong sRNA. This was experimentally validated by using either Lacl or tetR.
Additional Information
1) MATLAB Code
k3 = 1;
k3r = 50;
k4 = 50;
k5 = 700;
k6 = 10;
k7 = 110;
k8 = 700;
k9 = 10;
k10 = 6;
k11 = 2;
k11r = 70;
k12 = 1;
k13 = 2.5;
xyls = 1;
dt = 0.01;X = zeros(1000,8);
pm = 0.1;
pmx = 0.1;
mrna = 0.1;
lacl = 0.1;
pa = 0.1;
pal = 0.1;
srna = 0.1;
gfp = 0.1;
for i = 1:1000
Dpm = -k3*xyls*pm + k3r*pmx;
Dpmx = k3*xyls*pm - k3r*pmx;
Drna = k4*pm*pmx -k6*mrna - k10*srna*mrna;
Dlacl = k7*mrna +k11r*pal - k11*pa - k12*lacl;
Dpa = -k11*pal*lacl + k11r*pal;
Dpal = k11*pa*lacl - k11r*pal;
Dsrna = k8*pa - k10*srna*mrna - k9*srna;
Dgfp = k7*mrna - k13*gfp;pm = pm+Dpm*dt;
pmx = pmx+Dpmx*dt;
mrna = mrna+Drna*dt;
pmx = pmx+Dpmx*dt;
lacl= lacl+Dlacl*dt;
pa = pa+Dpa*dt;
pal = pal+Dpal*dt;
srna = srna+Dsrna*dt;
gfp = gfp+Dgfp*dt;
X(i,:) = [pm, pmx, mrna, lacl, pa, pal, srna, gfp];
end
Where the negative sign denotes the reaction rate of reverse reaction
2) Initial Values
a. First Bi-stable System
P = 1233
TF = 1273
I = 100
mrna = 300
R1 = 3
Apm2 = 4.5
pm2r1 = 3090
srna = 333
goi1 = 2
b. Second Bi-stable System
P2 = 89
TF2 = 95
I2 = 7
mrna2 = 3
R2 = 3
pm3 = 2
pm3r2 = 3
srna2 = 1.5
goi2 = 3
When the addition of inducer in the first bi-stable system triggers the functionality of recombinase, inducer in the second system is activated as time elapses. Thereby, the expression of toxin will be explicit at the end of the process
Reference
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