【introduction】

To be more user-friendly, 4# circuit contains a reporting system. After melting in water, the spores germinate and express blue pigment protein to report the best using time.
Meanwhile, 4# circuit could also ensure biosafety. Because other circuit do not have self-killing device, 4# engineering bacterial should kill all the bacterial after using.

Results

①

B.subtilis Plasmid PHT43+p43+amilCP
Colony PCR 14 single clones

【improving】

In our experiment , we discovered that the promoter P43 is too weak to express amilCP for a blue-color display. So we redesigned the circuits , aiming to find a more feasible plan, and test the part amilCP simultaneously. The new disign contains two circuits:

1 Pgrac-amilCP-Terminator

We use the Promoter grac , a promotor with lac promotor ,on the PHT vector to express amilCP, instead of Promoter 43. The Promoter grac is strong enough ,so we can easily see the results theoretically.

pgrac-amilCP overlap PCR（Right Three） PSBC3（Left Four ）

2 P43-amilcp-SigB-Terminator

We plan to use a positive feedback to magnify the expressing of amilCP. Because the P43 is a sigma B factor binding promotor, we designed a circuit, that the P43 is fused to the sigma B factor. We hope this could increase the response of P43.
Course of time limitation, we haven’t put this design to experiment.

【designing of the suicide system】

We design a circuit of killing switch based on its endogenous genetic system.
In B.subtilis, when it comes to the stationary phase, the environmental pressure increases and nutrition becomes limited, so B begin to produce spores. Now the community will be divided into two different parts. One of them are trying to kill others to get enough nutrient , delaying the production of spores and achieving a competitive advantage. Killing is mediated by the exported toxic protein SdpC. SdpI will appear on the membrane surface to avoid itself from being damaged. SdpI could bind free SdpC and autopressor SdpR, to remove SdpR’s inhibition against I and R, to produce more SdpI to offset SdpC, finally guaranteeing the subgroup alive, thereby delaying the spores production.

We transfer SdpC which is fused by promoter SdpI/R into high copy plasmids in order to damage the balance of the system, thereby killing whole colony. When SdpC appears, SdpI on the membrane will bind free SdpC and adsorb SdpR to cease its inhibition against SdpI P/R, trying to produce more SdpI. At the same time, it will activate the promoter SdpR/I in our circuits and generate more SdpC.The system would fall into an infinite loop, and according to our modeling ,the amount of SdpC increases beyond the ability of SdpI.Thus,the cells with protection mechanism will crack and die because of too much SdpC. All above forms the killing device. We Also designed a test circuit,which contains promotor grac and sdpABC only,aiming to determine the ability of SdpC.

Results

Colony PCR E.coli PHT43 + Promotor grac + SdpABC

PHT43 + Promotor SdpRI + SdpABC Enzyme digestion

There are both positive and negative feedback loops in this process. On the one hand, SdpI is unable to sequestrate the autorepressor, SdpR, until it captures the toxin, SdpC. The accumulation of SdpC will thus facilitate SdpI to capture more SdpR and thereby relieve the repression of SdpR, stimulating the expression of itself. This is the positive feedback loop which leads to the increasing accumulation of SdpC and finally the death of the bacteria. On the other hand, the removal of SdpR also enhance the expression of SdpI and accelerate the sequestration of SdpC, which forms a negative feedback loop whose effects contradict the positive feedback loop. However, since the copy number of SdpC is much higher, it is believed that the positive loop is strong enough to outweigh the negative one, which guarantees this mechanism will finally leads to collapse instead of equilibrium.

【References】

Parallel pathways of repression and antirepression governing the transition to stationary phase in Bacillus subtilis AV Banse, A Chastanet, L Rahn-Lee…,PNAS ,2008