Difference between revisions of "Part:BBa K557001"
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K557001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K557001 SequenceAndFeatures</partinfo>
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Background
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== Model ==
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This part is composed of a theophylline-sensitive aptamer,which will pair the ribosome binding site of the downstream gene in the absence of theophylline and thus inhibit the translation of the gene while expose the ribosome binding site in the presence of theophylline, and the CheZ gene, which is a phosphatase which dephosphorylates CheY-P and causes the flagellum to rotate counterclockwise and is essential for keeping the motility of E.coli. Thus this part provides a ligand-inducible motility and makes it possible to guide E.coli by theophylline.
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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 [http://2011.igem.org/Team:USTC-China/Project our system] to act as the main power to drive two parts of a bacterial colony away from each other.
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[[Image:Aptamer-toggleswitch.PNG]]<br/>
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[[Image:Aptamer-toggleswitch.PNG|center]]<br/>
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Figure 1: Model for how the theophylline-sensitive synthetic riboswitch controls the translation of the CheZ protein. In the absence of theophylline (left),the mRNA adopts a conformation in which the ribosome binding site is paired and translation of CheZ is inhibited. In the absence of CheZ, the protein CheY-P remains phosphorylated and the cells tumble in place. In the presence of theophylline (shown in red), the mRNA can adopt a conformation in which the ribosome binding site is exposed and CheZ is expressed, thus allowing the cells to run and tumble. (Images from: Shana Topp, Justin P. Gallivan (2006) Guiding Bacteria with Small Molecules and RNA. ''J.Am.Chem.Soc'', 129,6870-6811)
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== Experimental Data ==
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'''''Dose-Dependent Migration Ability of Aptamer-cheZ Device'''''<br/>
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To test the migration ability, selective media (tryptone broth with different ratio of agar(0.25%, 0.3%, 0.4%), 50μg/mL ampicllin, and various concentrations of theophylline(0mM, 0.25mM, 0.5mM, 0.5mM, 0.75mM, 1mM)) was prepared in Petri dishes(85mm dia). Diluted cell suspensions from mid-log-phase cultures were applied to the center of the plates, which were dried at room temperature at for 15 min, and incubated at 37°C for 10h, and the migration radii were determined by measuring the diameter of the outermost ring of growth. <br/>
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[[Image:A().png]]
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[[Image:A(1).png]]
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[[Image:A(2).png]]<br/>
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Figure2.((a)0.25%agar, (b)0.3%agar, (c)0.4%agar)<br/>
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'''The results(Figure2 & 3.) show that the Aptamer-cheZ Device is functional, and the 0.3%agar is the best ratio for the migration experiments.''''
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[[Image:X().jpg]]<br/>
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Figure3. The growing state of the reprogrammed bacteria with Aptamer-cheZ part(Left:0.3%agar with 0mM Thephylline, Right:0.3%agar with 0.25mM Thephylline)
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== Acknowledgements ==
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Here we want really want to thank the Professor J. S. Parkinson for providing ΔcheZ strain RP1616 and the corresponding wild type RP437 and Professor J. P. Gallivan for directing our construction of riboswitches.
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<br/>
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== Reference ==
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1.Shana Topp, Justin P. Gallivan (2006) Guiding Bacteria with Small Molecules and RNA. ''J.Am.Chem.Soc'', 129,6870-6811
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K557001 parameters</partinfo>
 
<partinfo>BBa_K557001 parameters</partinfo>
 
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Latest revision as of 16:32, 5 October 2011

Aptamer-CheZ: theophylline-sensitive synthetic riboswitch+phosphatase which dephosphorylates CheY-P


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Model

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 [http://2011.igem.org/Team:USTC-China/Project our system] to act as the main power to drive two parts of a bacterial colony away from each other.

Aptamer-toggleswitch.PNG

Figure 1: Model for how the theophylline-sensitive synthetic riboswitch controls the translation of the CheZ protein. In the absence of theophylline (left),the mRNA adopts a conformation in which the ribosome binding site is paired and translation of CheZ is inhibited. In the absence of CheZ, the protein CheY-P remains phosphorylated and the cells tumble in place. In the presence of theophylline (shown in red), the mRNA can adopt a conformation in which the ribosome binding site is exposed and CheZ is expressed, thus allowing the cells to run and tumble. (Images from: Shana Topp, Justin P. Gallivan (2006) Guiding Bacteria with Small Molecules and RNA. J.Am.Chem.Soc, 129,6870-6811)

Experimental Data

Dose-Dependent Migration Ability of Aptamer-cheZ Device
To test the migration ability, selective media (tryptone broth with different ratio of agar(0.25%, 0.3%, 0.4%), 50μg/mL ampicllin, and various concentrations of theophylline(0mM, 0.25mM, 0.5mM, 0.5mM, 0.75mM, 1mM)) was prepared in Petri dishes(85mm dia). Diluted cell suspensions from mid-log-phase cultures were applied to the center of the plates, which were dried at room temperature at for 15 min, and incubated at 37°C for 10h, and the migration radii were determined by measuring the diameter of the outermost ring of growth.
A().png A(1).png A(2).png
Figure2.((a)0.25%agar, (b)0.3%agar, (c)0.4%agar)
The results(Figure2 & 3.) show that the Aptamer-cheZ Device is functional, and the 0.3%agar is the best ratio for the migration experiments.' X().jpg
Figure3. The growing state of the reprogrammed bacteria with Aptamer-cheZ part(Left:0.3%agar with 0mM Thephylline, Right:0.3%agar with 0.25mM Thephylline)

Acknowledgements

Here we want really want to thank the Professor J. S. Parkinson for providing ΔcheZ strain RP1616 and the corresponding wild type RP437 and Professor J. P. Gallivan for directing our construction of riboswitches.

Reference

1.Shana Topp, Justin P. Gallivan (2006) Guiding Bacteria with Small Molecules and RNA. J.Am.Chem.Soc, 129,6870-6811