【Why Do We Design This Experiment】
Bacillus subtilis has been widely applied as engineered bacteria, especially in food industry and pharmaceutical industry, for its safety and excellent secretion capacity. Therefore, after comparing characters of distinct mutants we selected Bacillus subtilis WB800N mutant as our engineered bacteria and looked up plenty of papers to select the optimal conditions for our experiment. To our disappointment, very few experiments have been done on WB800N mutant, and most optimization experiments regarding Bacillus subtilis focus solely on the optimization of production of specific proteins produced by Bacillus subtilis. Consider the final goal of our project, it is imperative to design this experiment on our own to find out the best condition for Bacillus subtilis WB800N.
【Methodology】
Any optimization designs will inevitably involve the ideology of Design of Experiment (DOE), which includes several dependent plots. Among them Orthogonal Design and Response Surface Design, RSM for short, are the most common two in biological experiments. Generally, Orthogonal Design consumes less time and has been used more widely, yet it is not logically rigorous in mathematics, and sometimes it overlooks interactions and alias between or among factors. In contrast, RSM is constructed on rigorous mathematical theories and excels in data analysis. Having weighing the features of the two methods carefully, we finally chose RSM.
Sweeping Factors
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https://static.igem.org/mediawiki/2013/4/4d/Reporter_1.tif
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.
https://static.igem.org/mediawiki/2013/2/22/Reporter_2.tif
pgrac-amilCP overlap PCR（右三道） PSBC3双酶切（左四道 ）
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.
https://static.igem.org/mediawiki/2013/2/2b/Reporter_3.png
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
https://static.igem.org/mediawiki/2013/e/ec/Reporter_4.tif
Colony PCR E.coli PHT43 + Promotor grac + SdpABC
https://static.igem.org/mediawiki/2013/9/92/Reporter_5.tif
PHT43 + Promotor SdpRI + SdpABC Enzyme digestion
【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
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