Team:UFSCar-Brasil/killswitch.html
Kill Switch
How to kill them!?
The concern of a biosafety system was proposed to answer the social guidelines that followed the development of this project.
The adequation to a biosafety system for the product is characterized by the use of assassin genes that are repressed by a compound that does not affect neither the efficiency of our product nor human health. Thinking of it, it was observed that zinc would be a good compound to fulfill this task, since it does not pose any risks to human skin on low concentrations (CROSS et al., 2007).
Briefly, the KillSwitch is based on an operon present in Escherichia coli, the znuABC (PATZER & HANTKE, 1998). This operon is composed by three genes that are responsible for zinc uptake to intracellular space through the transcription of a transporter of ABC type. In this way, when there is zinc in intracellular space, it binds to a factor known as Zur, which inhibits the action of the operon interacting with its promoter region (JACKSON et al., 2008).
Using the mechanism of action from znuABC operon, and knowing the sequence of the three genes responsible for its functioning, the idea of our killswitch is based on the cloning of the promoter region of the operon and its use to induce the expression of assassin genes. With the assistance of PePPER, the promoter sequence of the operon was found. We obtained sequences that proved the promoter bidirectionality, and could isolate the sense and antisense sequences. Due the requirements of this project, it was not cloned completely, promoting only in one sense of direction.
Knowing the sequence of the protein factor Zur (PATZER & HANTKE, 1998), it was suggested the overexpression of it, by a constitutive promoter, to guarantee its presence in cell after the process of plasmolysis. Therefore, the demand of the factor necessary to silence the expression of the assassin genes and znuABC operon would be sufficed, retarding the biosafety system and ensuring the repellent action and duration.
After the interaction of the repellent with the skin, and the start of the expression of limonene synthase by osmotic shock, the bacteria will have its metabolism activated. It will be sued a protein responsible to absorb zinc, SmtA (YAMANAKA et al., 1995); initiated by the promoter pUspA previously described. In this way, the production will start by an osmotic stress caused by the contact of PEG with water present in human sweat on skin. With the performance of zinc transporters, from znuABC operon, and SmtA, the zinc concentration will decrease. Thus, the assassin genes, described next, when activated, promote cellular death.
Figure 1: Kill switch general mechanism.
Figure 2: Project module III comprising the partial circuit to Kill Switch as conceived.
KillerRed
KillerRed is a chromoprotein responsive to luminous irradiation which produces oxygen reactive species (LIAO et al., 2014). Its activity is related to \(O^{2-}\) superoxide production; which can react with amino acid lateral chains, lipids, DNA and RNA. It is important to highlight that this protein does not act before a luminous irradiation event occur (BULINA et al., 2006).
Barnase
Barnase is a bacterial protein with ribonuclease activity. It is naturally synthetized and secreted by the bacteria Bacillus amyloliquafaciens, however, it shows intense lethality when it is not bound to its inhibitor, Barstar (BUCKLE et al., 1994).
References
CROSS S. E., INNES B., ROBERTS M. S., TSUZUKI T., ROBERTSON T. A., MCCORMICK P. Human skin penetration of sunscreen nanoparticles: in-vitro assessment of a novel micronized zinc oxide formulation. Skin Pharmacol Physiol. 2007;20(3):148-54. Epub 17 Jan 2007;
PATZER, S. I.; HANTKE, K. The ZnuABC hight-affinity zinc uptake system and its regulator Zur in Escherichia coli. Molecular Microbiology. 1998.
YAMANAKA, K.; OGURA, T.; NIKI, H.. HIRAGA, S. Characterization of the smtA gene encoding an S-adenosylmethionine-dependent methyltransferase of Escherichia coli. FEMS Microbiol Lett. 1995.
LIAO, Z. X.; LI, Y. C.; LU, H. M.; SUNG, H. W. A genetically-encoded KillerRed protein as an intrinsically generated photosensitizer for photodynamic therapy. Biomaterials. 2014.
BULINA, M. E.; CHUDAKOV, D. M.; BRITANOVA, O. V.; YANUSHEVICH, Y. G.; STAROVEROV, D. B.; CHEPURNYKH, T. V.; MERZLYAK, E. M.; SHKROB, M. A.; LUKYANOV, S.; LUKYANOV, K. A. A genetically encoded photosensitizer. Nat Biotechnol. 2006.
BUCKLE AM, SCHREIBER G, FERSHT AR. Protein-protein recognition: crystal structural analysis of a barnase-barstar complex at 2.0-A resolution. Biochemistry 33(30): 8878–89. 1994.
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