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The Synchroscillator: A Tunable Synchronized Oscillatory System

This BioBrick device yields an oscillating pattern of Green Fluorescent Protein output, in the way described below. It contains two differing hybrid promoters that allow for stabilization of the oscillation.

Usage and Biology

The Tunable Syncrhonized Oscillatory System (Synchroscillator) consists mainly of one negative feedback loop, i.e. a biological process that downregulates itself, one positive feedback loop, i.e. a biological process that induces itself, and the reporter protein Green Fluorescent Protein (GFP) (with an LVA Tag). Both feedback loops are regulated by hybrid promoters that can decrease the transcription rates upon presence of the Tet Repressor Protein (TetR) or lac repressor (LacI) DNA-binding protein.

The positive feedback loop is created by the quorum sensing (QS) molecule, or autoinducer: 3OC6HSL. This autoinducer is able to diffuse through a colony of bacteria (and was found in a Vibrio species that uses it to react to its cell density in a culture). 3OC6HSL is produced by the enzyme encoded by the luxI gene. Most important of this is that 3OC6HSL is able to increase the transcription rate of the luxI gene, in the whole colony. It does so, by binding to the hybrid 3OC6HSL-LacI promoter if LacI is absent.

Interacting with the positive feedback loop is the negative feedback loop. Also this feedback loop is caused by presence of the QS molecule. That is, 3OC6HSL binds a (hybrid) promoter as a complex with the constitutively expressed LuxR protein. This complex raises the transcription rate of the autoinducer inactivation enzyme A (aiiA) gene and therefore the concentration AiiA. As its gene name suggests, the AiiA is capable of inactivating the 3OC6HSL autoinducer. The promoter in front of the aiiA gene is a hybrid promoter and can also be repressed by the Tet Repressor Protein (TetR). In conclusion, the QS autoinducer is both formed and degraded by the BioBrick system and both processes can be set by LacI and TetR initially.

If the concentration rise and drop of 3OC6HSL occurred with the same rate by both the positive and negative feedback loops, the concentration of the QS molecule would remain constant. However, under the right circumstances an oscillation could be created. When the autoinducer concentration rises before it reaches a certain threshold to initiate the negative feedback loop, the GFP signal, which depends on the autoinducer concentration, would first increase and then remain constant. However, the production and degradation rate of 3OC6HSL are not the same. AiiA inactivates 3OC6HSL more quickly than it is formed. The negative feedback loop causes that after a while aiiA is no longer expressed. After this, the concentration will still be be lowered until too much AiiA is deteriorated, which occurs in a natural process inside the cell. Nonetheless, the positive feedback loop is still active and will increase the autoinducer formation speed again and start the next cycle.

More about this BioBrick device? Another [http://2011.igem.org/Team:Wageningen_UR/Project/IntroductionProj1 description] and the [http://2011.igem.org/Team:Wageningen_UR/Project/ModelingProj1#Mathematical_model_of_the_construct modeling] of are placed on the Wiki page of the project it was made in.

Experimental data

The tunable oscillator was tested in a flow chamber that contained a microdish, designed by Team Wageningen UR 2011. Every 10 min. pictures were taken with an Olympus fluorescence microscope. The setup of the flow chamber is explained [http://2011.igem.org/Team:Wageningen_UR/Project/DevicesSetup here]. After an overnight incubation in the microdish a series of pictures was taken. This was also done for E. coli transformed with ptet-GFP as a fluorescent negative control for oscillation. Four wells from the microdish were clearly visible and those were analyzed using the program [http://rsbweb.nih.gov/ij/ ImageJ]. The outcome is presented in the graph below.

ptet-GFP shows a stable expression over time. This confirms that the differences in fluorescence is not due to fluctuations of the Xenon lamp. ptet-GFP shows a stable expression over time. This confirms that the differences in fluorescence is not due to fluctuations of the Xenon lamp. The Tunable Oscillator was ran on two different dates, each time four wells from the microdish were analyzed with ImageJ. Clearly visible are the large fluctuations in fluorescence. This is the case for both measurement series that were done for the Tunable Oscillator. Especially in the beginning there seems to be an oscillation that looks stable. Both series seem to have the stable periods, and they coincide. After a few hours, however, the oscillation is disrupted and periods are hardly distinguishable.

For an impression of the Tunable in the microdish, see below. For more information on the device and circuitry, visit our [http://2011.igem.org/Team:Wageningen_UR wiki].

Safety Aspects

A number of safety questions regarding the BioBrick system's [http://2011.igem.org/Team:Wageningen_UR/Safety/One#Risk_Identification_of_BioBrick_System_Inside_the_Cell_Chassis biological safety] and [http://2011.igem.org/Team:Wageningen_UR/Safety/Five biological security] have been answered, you can read specific answers by following the links.

Sequence and Features