Difference between revisions of "Part:BBa K546546"

Revision as of 19:32, 21 September 2011

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

This system consists of a positive and a negative feedback loop. The positive feedback loop is initiated when the constitutively expressed luxR forms a complex with AHL- which is produced by the protein LuxI. The complex up-regulates the expression of luxI, GFP and AiiA, at first exponentially increasing LuxI, GFP and AiiA levels because the maturation time of LuxI is shorter than that of AiiA. Then the negative feedback loop gets the upper hand: Once maturated, the enzyme AiiA degrades AHL faster than luxI can produce it. Because GFP is under the control of the same promoter as luxI it is highly expressed until AHL is rapidly being degraded by AiiA. Once the AHL concentration is lowered enough, the expression of GFP is halted. At this point the net change in GFP will thus become negative due to the degradation. Since AiiA expression is initiated by the luxR-AHL complex, the production of AiiA also halts which causes the net change of AiiA to become negative and this eventually causes the concentration of AiiA to become low enough to allow a net production of AHL again. Since AHL is a quorum sensing molecule all cells are synchronised in their oscillatory behavior.

Furthermore, this system implements two hybrid promoters: One controlling the transcriptional rate of luxI and the other one controlling the transcriptional rate of AiiA. These two promoters are respectively repressible by IPTG and aTc. Therefore, it should be possible to tune the period and the amplitude of the synchronized oscillations.

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 2781
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1184
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 985
Illegal BsaI.rc site found at 2042
Illegal BsaI.rc site found at 2930
Illegal BsaI.rc site found at 3657

@@ Line 5: / Line 5: @@
 ===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.
+This system consists of a positive and a negative feedback loop. The positive feedback loop is initiated when the constitutively expressed luxR forms a complex with AHL- which is produced by the protein LuxI. The complex  up-regulates the expression of luxI, GFP and AiiA, at first exponentially increasing LuxI, GFP and AiiA levels because the maturation time of LuxI is shorter than that of AiiA. Then the negative feedback loop gets the upper hand: Once maturated, the enzyme AiiA degrades AHL faster than luxI can produce it. Because GFP is under the control of the same promoter as luxI it is highly expressed until AHL is rapidly being degraded by AiiA. Once the AHL concentration is lowered enough, the expression of GFP is halted. At this point the net change in GFP will thus become negative due to the degradation. Since AiiA expression is initiated by the luxR-AHL complex, the production of AiiA also halts which causes the net change of AiiA to become negative and this eventually causes the concentration of AiiA to become low enough to allow a net production of AHL again. Since AHL is a quorum sensing molecule all cells are synchronised in their oscillatory behavior.
-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.
+Furthermore, this system implements two hybrid promoters: One controlling the transcriptional rate of luxI and the other one controlling the transcriptional rate of AiiA. These two promoters are respectively repressible by IPTG and aTc. Therefore, it should be possible to tune the period and the amplitude of the synchronized oscillations.
-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==