Team:Freiburg/Killswitch
Kill switch
Before our Nanocillus can be used as a target drug delivery chassis, adequate safety precautions have to be taken. The most obvious is certainly to prevent Nanocillus to re-enter the vegetative state.The Munich iGEM Team 2012 tried to implement a kill switch for B.subtilis. Their system composes of two components. The first step of the system contains an alternative sigma factor called ecf41 Bli aa1-204 1which is activated by sigma factor G by the sigma G sensitive promotor P spoIVZ2.
Sigma factor G is a transcriptional regulator which is activated at a late stage during the germination process of spores. The transcriptional regulator activates the second step the the kill swith leading to the expression and production of a toxin.
The production of cell toxin, at this time point, prevents the spores from re-entering in the vegetative circuit. While the concentration of the alternative sigma factor ecf41 Bli aa1-204 rises, it is activating an unique target promoter called P ydfG. This leads to the expression of MazF, a toxin that degrades mRNA.
Figure 1: Kill switch of Munich 2012
The Munich iGEM Team 2012 had the problem of overproduction of the toxin, already during their cloning procedure of the BioBrick part leading to cell death of E.coli. To overcome this problem, we thought about using a different toxin. Following intensive literature research we came up with the idea of using the T4-Holin kill switch.
Unlike mazF this toxin needs a certain threshold and a higher concentration of proteins to become toxic, we decided to create a simple model describing our kill switch to validate if our system would theoretically work. The following paragraph describes the single steps. Step 1: (alternative sigma factor ecf41 Bli aa1-204 protein expression activated by sigma factor G)
Figure 2: (sigma factor G)
{bk1=constant for an approximation of the increase of sigma factor G, bk2=Protein degradation rate, b[t]=total amount of sigma factor G protein, db/dt=change of the total amount of sigma factor G protein in a certain time interval}
Figure 3: (sigma factor ecf41 Bli aa1-204 mRNA)
{dx/dt=change of the total amount of sigma factor ecf41 Bli aa1-204 protein mRNA in a certain time interval, xv1=DNA concentration per cell ,k11=leaky expression ,k21=expression constant, a=activator concentration ,n=Hill coefficient, xv2=DNA concentration per cell ,xK=activation constant, k41=mRNA degradation rate ,x[t]=total amount of sigma factor ecf41 Bli aa1-204 protein mRNA}
Figure 4: (sigma factor ecf41 Bli aa1-204 protein)
{yk1=mRNA translation rate, yk2=protein degradation rate, y[t]=total amount of sigma factor ecf41 Bli aa1-204 protein, dy/dt=change of the total amount of sigma factor ecf41 Bli aa1-204 protein in a certain time interval} Step 2: (T4-Holin protein expression activated by sigma factor ecf41 Bli aa1-204)
Figure 5: (T4-Holin mRNA)
{dz/dt=change of the total amount of T4-Holin protein mRNA in a certain time interval, v=DNA concentration per cell ,k11=leaky expression ,k21=expression constant, a=activator concentration ,n=Hill coefficient ,zK=activation constant, k41=mRNA degradation rate ,z[t]=total amount of T4-Holin protein mRNA}
Figure 6: (T4-Holin protein)
{ak1=mRNA translation rate,ak2=protein degradation rate,a[t]=total amount of T4-Holin protein, da/dt=change of the total amount of T4-Holin protein in a certain time interval}
Figure 7: Increase of T4-Holin over time.
{xv1=1, xk1=0.3, xv2 =1, xk2=0.9, xK= 2.1, xk3=0.3, yk1=0.1, yk2=.2, zv1=4, zk1=0.6, zv2=0.7,zk2= 12, zK=0.4, zk3=0.35, ak1=0.2, ak2=0.1, bk1=0.12, bk2=0.012, n=1, time in hours, concentration in nM}
But this is not all. During our literature research we cam across with the mazE/F system, a toxin/antitoxin system. By studying more about this system and looking for an improved kill switch system, We came up with idea to combine the mazE/F system with a mazE anti-sense mRNA.
Figure 8: The mazE/F system3. This would cause a fast decrease of mazE and would allow mazF to unfold its toxic effect4.
Figure 7: Kill switch containing mazE anti sense mRNA.
With the help of he Zurich iGEM Team 2016, we calculated the single parameter for our system
Modeling Collaboration with Zürich
[1]=Amitai, S., Yassin, Y., & Engelberg-Kulka, H., MazF-mediated cell death in Escherichia coli: a point of no return, 2004, Journal of bacteriology, 186(24), 8295-8300.
[2]=Steil, L., Serrano, M., Henriques, A. O., & Völker, U, Genome-wide analysis of temporally regulated and compartment-specific gene expression in sporulating cells of Bacillus subtilis, 2005, Microbiology, 151(2), 399-420.
[3]=Wecke, T., Halang, P., Staroń, A., Dufour, Y. S., Donohue, T. J., & Mascher, T., Extracytoplasmic function σ factors of the widely distributed group ECF41 contain a fused regulatory domain, 2012, Microbiologyopen, 1(2), 194-213.
[4]=Yamaguchi, Y., Inouye, M., The multiple levels of autoregulation of the MazF–MazE toxin–antitoxin system., 2011, Nature Reviews Microbiology 9, 779-790
[2]=Steil, L., Serrano, M., Henriques, A. O., & Völker, U, Genome-wide analysis of temporally regulated and compartment-specific gene expression in sporulating cells of Bacillus subtilis, 2005, Microbiology, 151(2), 399-420.
[3]=Wecke, T., Halang, P., Staroń, A., Dufour, Y. S., Donohue, T. J., & Mascher, T., Extracytoplasmic function σ factors of the widely distributed group ECF41 contain a fused regulatory domain, 2012, Microbiologyopen, 1(2), 194-213.
[4]=Yamaguchi, Y., Inouye, M., The multiple levels of autoregulation of the MazF–MazE toxin–antitoxin system., 2011, Nature Reviews Microbiology 9, 779-790
