Team:BNU-CHINA/Sample
Switch
When $a \ne 0$, there are two solutions to \(ax^2 + bx + c = 0\) and they are $$x = {-b \pm \sqrt{b^2-4ac} \over 2a}.$$In this part, we designed a bi-directional transcription system which is regulated by light. It can express toxicity or attractant in different conditions. Specifically, we linked poison protein genes and attractant gene to different sites of reversible promoter(Pcon). This reversible promoter can change the direction with the action of int protein, whose expression is regulated by PompR, a promoter regulated by light. To be specific, an enzyme encoded by ho1 gene can catalyze the synthesis of BV protein, which can catalyze another protein which is encoded by pcya gene to synthesize PCB protein. PCB protein can combine with cph8 protein and form cph8-PCB complex. This complex is easy to be phosphorylated under normal conditions. The result of phosphorylation is ompR protein can segregate from the complex and combine with Pompr and promote the expression of int protein. The specific numbers of biobricks are listed at the Design part of the Project.
The mechanism of this system is that in the condition of light, cph8-PCB complex cannot be phosphorylated, correspondingly, ompR protein cannot combine with PompR, so int protein is fail to be synthesized.
So, our system’s mechanism of action is that we use light with certain wavelength to irradiate the system in initial, in order to inhibit the expression of int protein and on the contrary, express the attractant. When the attractant accumulate to a certain concentration, we will move away the source of light, in order to express int protein, so that the system can transcribe in the opposite direction to express poison protein. The specific mechanism of this system is shown in the figure below. (Figure 1)
In this system, we can regulate the expression degree through the bi-directional transcription mechanism which is regulated by light. The meaning of this system is that we can avoid the environmental or biological hazards caused by the high expression of poison protein and attractant at the same time.
Suicide
We not only considered the potential safety hazard caused by the high expression of poison protein and attractant, but also designed a suicide system for our engineering bacteria, in order to prevent its spread in the environment and mass propagation.
The mechanism of our suicide switch is that engineering bacteria is sensitive to the population size. Our system contains a section of luxI gene, and a synthase encoded by it is able to catalyze the synthesis of AHL molecule. AHL is a kind of organic small molecule which is able to across the membrane freely. Another gene contained in our system called LuxR, it encodes LuxR protein, which can combine with AHL molecule. When AHL molecule reaches a certain concentration, LuxR protein will combine with PluxR promoter, and activate the expression of MazF poison protein, thus make our bacteria suicide themselves. The specific numbers of biobricks are listed at the Module3 part of the Project.
We can see that the quantity of bacteria can maintain in a relatively stable condition through this system, in order to limit the propagation of our engineering bacteria which have antibiotic resistance.
Lab&Environment safety
We have a strict safety regulations in our laboratory. The rules are as follows. When we enter the laboratory, all the members are required to wear the lab-gown and the latex examination gloves. During our experiment, such as the bacterination, we have to do it in the Clean Bench. And when we have to use the hazardous reagents, we must do it in the fuming cupboard as the safety regulations told. Moreover, the reagents we used should be poured into disposal bottle. And the mediums must be throw after doing the ultra-high-temperature sterilizing. In addition, the trash must be classified to throw in the normal trash can or the medical waste can.
As for the environment safety, we also considerate how our engineering bacteria works. We plan to design a semienclosed device to cultivate engineering bacteria to trap and kill the nematodes by modeling. So it avoids the security risk of direct contact of the soil and the bacteria. Furthermore, as we said before, we designed the suicide system to avoid the excess propagation of the recombinant bacteria.