Difference between revisions of "Part:BBa K546546"
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<partinfo>BBa_K546546 short</partinfo>
 
<partinfo>BBa_K546546 short</partinfo>
  
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This BioBrick device yields an oscillating pattern of GFP (with an LVA Tag) expression, in the way described below. It contains two differing hybrid promoters that allows for stabilization of the oscillation.
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===Usage and Biology===
 
===Usage and Biology===
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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). 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.
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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.
  
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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.
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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.
  
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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.
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For a detailed explanation about The Synchroscillator click [http://2011.igem.org/Team:Wageningen_UR/Project/IntroductionProj1 here].
  
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If the concentration rise and drop of 3OC6HSL occured 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 finally initiates the negative feedback loop the GFP signal, that depends on the autoinducer concentration, would first increase and remain constant. However, the the production and degradation rate of 3OC6HSL are not the same. AiiA inactivates 3OC6HSL more quickly than it's formed. After the concentration of the autoinducer has dropped so much that aiiA no longer is expressed the concentration will still be be lowered until too much AiiA is deteriorated, which is a natural process inside the cell. The positive feedback loop will increase the autoinducer production rate again and start the next cycle.
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==Experimental data==
  
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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.
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The tunable oscillator was tested in a flow chamber that contained a microdish, which was designed by Team Wageningen UR 2011. Every 10 minutes, a picture was taken with an Olympus fluorescence microscope. The set-up of the flow chamber is explained [http://2011.igem.org/Team:Wageningen_UR/Project/DevicesSetup here].
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After an overnight incubation in the microdish a series of pictures was taken. This was also done for <I>E. coli</I> transformed with ptet-GFP as a fluorescent negative control for oscillations. 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.
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[[Image: Synchroscillator_timelapse_study.png|center]]
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ptet-GFP shows a stable expression over time. This confirms that the differences in fluorescence are not due to fluctuations of the Xenon lamp. The Tunable Oscillator was run on two different dates and 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 at the start there seems to be an oscillation that looks stable. Both series seem to have the stable periods, which coincide. After a few hours, however, the oscillation is disrupted and periods are hardly distinguishable. 
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For an impression of the Synchroscillator in the microdish, see the picture below. For more information about the [http://2011.igem.org/Team:Wageningen_UR/Project/Devices device] and about the [http://2011.igem.org/Team:Wageningen_UR/Project/CompleteProject1Description circuitry], visit our wiki. 
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=== Model ===
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The double tunability of the Synchroscillator was modeled in Matlab. The resulting graphs are depicted below. They show the different behavior of LuxI, AiiA and internal/external AHL concentrations depending on the amount of IPTG and aTc present in the system.
 
   
 
   
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[[Image:Graph5_2_synchroscillator_WUR.png|450px]] [[Image:Graph4_2_synchroscillator_WUR.png|450px|right]]
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'''Fig.1 and 2:''' ''Different shapes of the oscillations depending on the proportion of IPTG and aTc.''
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[[Image:Model_parts.igem_WUR.png|450px|center]]
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'''Fig.3:''' ''Resulting oscillations when adding the same amount of IPTG and aTc''
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===Safety Aspects===
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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.
  
 
<span class='h3bb'>'''Sequence and Features'''</span>
 
<span class='h3bb'>'''Sequence and Features'''</span>

Latest revision as of 03:20, 22 September 2011

The Synchroscillator: A Tunable Synchronized Oscillatory System


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.

For a detailed explanation about The Synchroscillator click [http://2011.igem.org/Team:Wageningen_UR/Project/IntroductionProj1 here].

Experimental data

The tunable oscillator was tested in a flow chamber that contained a microdish, which was designed by Team Wageningen UR 2011. Every 10 minutes, a picture was taken with an Olympus fluorescence microscope. The set-up 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 oscillations. 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.


Synchroscillator timelapse study.png


ptet-GFP shows a stable expression over time. This confirms that the differences in fluorescence are not due to fluctuations of the Xenon lamp. The Tunable Oscillator was run on two different dates and 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 at the start there seems to be an oscillation that looks stable. Both series seem to have the stable periods, which coincide. After a few hours, however, the oscillation is disrupted and periods are hardly distinguishable.

For an impression of the Synchroscillator in the microdish, see the picture below. For more information about the [http://2011.igem.org/Team:Wageningen_UR/Project/Devices device] and about the [http://2011.igem.org/Team:Wageningen_UR/Project/CompleteProject1Description circuitry], visit our wiki.


Model

The double tunability of the Synchroscillator was modeled in Matlab. The resulting graphs are depicted below. They show the different behavior of LuxI, AiiA and internal/external AHL concentrations depending on the amount of IPTG and aTc present in the system.

Graph5 2 synchroscillator WUR.png
Graph4 2 synchroscillator WUR.png


Fig.1 and 2: Different shapes of the oscillations depending on the proportion of IPTG and aTc.

Model parts.igem WUR.png

Fig.3: Resulting oscillations when adding the same amount of IPTG and aTc

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