From 2007.igem.org

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Self-differentiated Bacterial Assembly Line

Our projects concerns with the ability for bacterial cells to differentiate out of homogeneous conditions to achieve the division of labor. We want to design devices conferring host cells with the ability to spontaneously form cooperating groups and to take consecutive step even when the genetic background and environmental inputs are identical. To break the mirror in such homogeneous condition, we need two devices respectively responsible for temporal and spatial differentiation. The implementation and application of such devices will lead to a picture of future bioengineering where complex programs consisted of sequential steps (structure oriented programs) and cooperating agencies (forked instances of a single class, object and event oriented) can be embedded in a single genome. The expression of such a genome pre-programmed both temporally and spatially both resembles the differentiation process of multicellular organism and a assembly plant built out of a blueprint.

Hop Count with Conjugation

Hop counting is a cell-cell communication system in which the signals transfered among the cells record the number of times of transfer. With this system, cells can use the transmitted signal as topological cues of their neighborhood, and start the first step of differentiation and cooperation.

We have adapted the natural conjugation systems to a communication device where the conjugative plasmids as the vector for the transfered signal. The numbering signals are directly encoded on the transmitted DNA sequence. Utilizing a special character of the rolling-cycle replication of DNA during conjugation, we use a design where independent tandem oriT (BBa_J01002 and more) pairs are inserted into the same plasmids, which lost one tandem region at one conjugation event. Thus the number of conjugation are recorded on the presence/absence of regions on the plasmid of arbitrary length . Sequential conjugation and deletion of the different oriT pairs can be achieved by fine control of the expression of the cis-acting elements of the independent conjugation systems. In fact, we inserted the enzymes directly responsible for the initiation of conjugative DNA replication (we called them relaxases) followed by a terminator between the corresponding oriTs. The expression of all the enzymes are under the control of a single promoter. The terminators ensure only the relaxase of the most upstream undeleted tandem oriT regions are expressed. This design ganrantee the independent conjugative systems are deleted at specified order.

Currently, we have tested and constructed three set of independent conjugative systems and validate the feasibility of our design with the F system. The next step is to do the 3-step conjugation-counting process in a single strain.

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Push-on-push-off Switch

Push-on-push-off switches are binary switches flipping to different direction under the same impulses, depending on the previous states it resides. It differed from a toggle switch that the forces flip the switch back and force are exactly the same. This allows the cells to do sequential logic utilizing both the information of present and pst events.

We have designed a gene regulatory circuit that senses Ultra-Violet light (UV) irradiation as input signal. Under intermittent irradiation, two reporter genes are to be induced alternately. The circuit can be divided into two associated parts: a bistable switch and a NOR gate. The bistable switch resembles a toggle switch where the NOR gate is controlled by the bistable switch and the input signal. On the other hand, the output of the NOR gate can switch the bistable state from one to the other. In this way, the NOR gate combined the information of present inputs and the previous states of the system, and thus flipping the switch in different directions based on the previous events, even though the present inputs are always the same.

Thus far, we have constructed and tested a series of NOR gates and one bistable switch, successfully obtaining the desired dynamics for the previous design(these dynamics may also be very useful to many other applications). The next is to combine the two together and tune the system to obtain the desired function.

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