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Bacterial ‘Amitosis’

So far we have successfully constructed a novel system in which bacterial colonies will automatically divide into two parts after certain time. Over the summer we have been working on assembling riboswitches finely tuned by small molecules, which will act as the main power to drive two parts away from each other, and toggle switches pushed on and off while memorizing current state, which will play the role of giving birth to two ‘different’ kinds of bacteria in one colony. Furthermore, we have been modulating the toggle switch to produce a more balanced ratio and creatively integrating quorum sensing into our system to optimize our results. As to modeling, we have not only been building models of the movement in this ‘amitosis’, but also been collecting and analyzing data for a aptamers database for small molecules and corresponding genomic sequences and structures in guiding bacteria. 1.Overview 1) Abstract So far we have successfully constructed a novel system in which bacterial colonies will automatically divide into two parts after certain time. Over the summer we have been working on assembling riboswitches finely tuned by small molecules, which will act as the main power to drive two parts away from each other, and toggle switches pushed on and off while memorizing current state, which will play the role of giving birth to two ‘different’ kinds of bacteria in one colony. Furthermore, we have been modulating the toggle switch to produce a more balanced ratio and creatively integrating quorum sensing system into our system to optimize our results. As to modeling, we have not only been building models of the movement but also been collecting and analyzing data for a aptamers databasefor small molecules and corresponding genomic sequences and structures in guiding bacteria. Finally, we are intended to construct a Artificial Innate Immunity System (AIIS) with our self-organized bacteria, in which reprogrammed intestinal bacteria is used as a safety weapon to destroy the pathogens which invade the intestinal mucosal system. 2) Introduction/Motivation Cells grow and cells divide. This is the basis of life and nature. In our eyes, a bacterial colony, which is also formed by bacterial growth and division, is a big cell too. Given that bacteria always play their roles as a ‘group’ instead of an ‘individual’, a bacterial colony can be considered as the unit of our intended function. It may not be hard to imagine if this unit could grow and divide itself automatically, significant applications would be developed. In fact, similar ideas have already been realized in eukaryotic systems. Chemotaxis has always been a powerful tool in organizing bacteria’s movement and it’s always an important ability to reprogram bacteria to follow entirely new chemical signals. Riboswitches, which have been a hot issue of synthetic biology recently, can be used to guide E.Coli toward and precisely localized to a completely new chemical signal. We take advantage of them in our system to act as the main power to drive two parts of a bacterial colony away from each other. Now the key problem is to produce two different kinds of bacterial in one colony, in which one kind could be guided by riboswitches mentioned above while the other would keep still or move to the opposite side. The work of PKU-IGEM 2007 provides us with a ideal platform in which bacterial are equipped with devices to differentiate out of homogeneous conditions. Thanks to their warm help, we acquire Toggle-switches and made efforts to modulate the switch into to produce a more balanced ratio, so that the results would be more likely to be apparently visible. With the help of modeling, it’s spotted that the part driven away cannot be efficiently turned into another colony compared with the other part staying, since bacteria tend to move instead of grouping on their way towards the most concentrated location of the chemical signal. Quorum sensing, as another most applied mechanism in synthetic biology, give us a hint in solving this problem. The final step is to picture the blueprint of the application of our system. Here we present one in medical area, in which reprogrammed intestinal bacteria can help you with pathogens invasion. Given the difficulties of human experiment, we conduct a simulation in E.coli. 3) Method a)Verification of ΔcheZ strains b)Construction of Riboswitch: Theophylline guided riboswitch + cheZ c)Verification of Toggle-switches d)Modulate Toggle-switches to produce more balanced ratio e)Incorporation of Riboswitch into one side of Toggle-switch f)Verification of the constructed system’s function g)Integration of Quorum-sensing system h)Test i)Application: simulation of AIIS 4) Result 5) Conclusion 6) Discussion 7) Limitations & Future Work 8) References