Difference between revisions of "Part:BBa K3267000"

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This part will respond to the electric potential via various redox modulators and the transformed cell begins to have the activated transcription factor, which is SoxR protein. This transcription factor protein binds to the bidirectional promoter region, then cells translate more SoxR and LuxI.
 
This part will respond to the electric potential via various redox modulators and the transformed cell begins to have the activated transcription factor, which is SoxR protein. This transcription factor protein binds to the bidirectional promoter region, then cells translate more SoxR and LuxI.
  
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===Biology===
+
==Biology==
 
The Relay part contains E.coli. intrinsic the bidirectional promoter region of the SoxRS regulon. The SoxR coding sequence is truncated and LuxI coding region is added followed by a ssRA tag. LuxI gene is originally from the bacterium A. fischeri and encodes Acyl-homoserine-lactose synthase. LuxI synthases Acyl-homoserine-lactose (AHL) which is known as autoinducing peptides. The peptides are released in the extracellular environment, and other cells are able to detect the change in concentration of this peptide. This autoinduction process is called quorum-sensing (QS).
 
The Relay part contains E.coli. intrinsic the bidirectional promoter region of the SoxRS regulon. The SoxR coding sequence is truncated and LuxI coding region is added followed by a ssRA tag. LuxI gene is originally from the bacterium A. fischeri and encodes Acyl-homoserine-lactose synthase. LuxI synthases Acyl-homoserine-lactose (AHL) which is known as autoinducing peptides. The peptides are released in the extracellular environment, and other cells are able to detect the change in concentration of this peptide. This autoinduction process is called quorum-sensing (QS).
  
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===Usage===
+
==Usage==
 
In our device, we use multiple redox modulators to convert electric stimuli into chemical signals. [Fig 1] The toxin of Gram-negative bacterium Pseudomonas aeruginosa, Pyocyanin is used as the initiator of this redox cycle. Added pyocyanin is the oxidized form and it oxidizes SoxR Transcription Factor protein to activate. The ubiquitous Menaquinone transports an electron across the membrane and Pyocyanin is again oxidized. Ferrocyanide/Ferricyanide in the extracellular environment gets reduced via Menaquinone and the positive potential oxidizes Ferrocyanide to keep modulators oxidized. Therefore, the cycle keeps activating SoxR protein. The activated SoxR protein binds to the pSoxR/pSoxS bidirectional promoter region and expresses LuxI and SoxR proteins. SoxR protein is again activated under the positive electric potential as explained above. LuxI protein acts as Acyl-homoserine lactone (AHL) synthase. AHL will be diffused outside the cell and induces the surrounding cells' response to the change in the concentration of AHL.
 
In our device, we use multiple redox modulators to convert electric stimuli into chemical signals. [Fig 1] The toxin of Gram-negative bacterium Pseudomonas aeruginosa, Pyocyanin is used as the initiator of this redox cycle. Added pyocyanin is the oxidized form and it oxidizes SoxR Transcription Factor protein to activate. The ubiquitous Menaquinone transports an electron across the membrane and Pyocyanin is again oxidized. Ferrocyanide/Ferricyanide in the extracellular environment gets reduced via Menaquinone and the positive potential oxidizes Ferrocyanide to keep modulators oxidized. Therefore, the cycle keeps activating SoxR protein. The activated SoxR protein binds to the pSoxR/pSoxS bidirectional promoter region and expresses LuxI and SoxR proteins. SoxR protein is again activated under the positive electric potential as explained above. LuxI protein acts as Acyl-homoserine lactone (AHL) synthase. AHL will be diffused outside the cell and induces the surrounding cells' response to the change in the concentration of AHL.
  
−
===Characterization===
+
 
 +
[[File:T--NYU_Shanghai--ResultsFig6.jpg|thumb|center|Figure 1: Quorum Sensing by Electric Stimulation, this part is on the top]]
 +
 
 +
==Characterization==
 
NYU Shanghai iGEM team characterized this part by applying electric stimulus directly onto the agar plate that has necessary electromodulators and transformed cells with this Biopart. As we have known that 2.5uM Pyocyanin and 5mM Ferrocyanide(R)/5mM Ferricyanide (O) optimizes the activation of pSoxRS regulons, we mixed these amount of drugs in the given agar to be solidified in the plates with the two holes with electrodes. (Electrodes are covered by the agar so that cell culture is not exposed to the graphite.) Sodium sulfite is also added to create anaerobic environment on the plate.
 
NYU Shanghai iGEM team characterized this part by applying electric stimulus directly onto the agar plate that has necessary electromodulators and transformed cells with this Biopart. As we have known that 2.5uM Pyocyanin and 5mM Ferrocyanide(R)/5mM Ferricyanide (O) optimizes the activation of pSoxRS regulons, we mixed these amount of drugs in the given agar to be solidified in the plates with the two holes with electrodes. (Electrodes are covered by the agar so that cell culture is not exposed to the graphite.) Sodium sulfite is also added to create anaerobic environment on the plate.
  
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[[File:T--NYU_Shanghai--ResultsFig7.jpg|thumb|center|Figure 2: GFP Expression Level of Co-culture of Relay and Receiver part, Individual culture of Relay and Receiver, and No Cells. Every sample is stimulated by 0.5V potential for an hour.]]
 
[[File:T--NYU_Shanghai--ResultsFig7.jpg|thumb|center|Figure 2: GFP Expression Level of Co-culture of Relay and Receiver part, Individual culture of Relay and Receiver, and No Cells. Every sample is stimulated by 0.5V potential for an hour.]]
  
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[[File:T--NYU_Shanghai--ResultsFig8.jpg|thumb|center|Figure 3: Quorum Sensing by Electric Stimulation]]
+
[[File:T--NYU_Shanghai--ResultsFig8.jpg|thumb|center|Figure 3: Quorum Sensing by Electric Stimulation- UV]]
 +
 
 +
The quantification shows that this part enhances the GFP expression level when it is used with the Receiver cell (BBa_K3267001) and the role of Relay cells is showing that there are no colonies glowing around the positive electrodes (Left, Fig 3). These areas are for the receiver cell that responds to the positive electric potential while surrounding colonies are Receiver part cells (BBa_K3267001) which responded to the AHL gradient.
  
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The quantification shows that this part enhances the GFP expression level when it is used with the Receiver cell (BBa_K3267001) and enables the quorum sensing as you see the positive electrode (Left, Fig 3) has more colonies of the E.coli. responding to the electric stimuli.
+
==Reference==
 +
T. Tschirhart, E. Kim, R. McKay, H. Ueda, H.C. Wu, A.E. Pottash, A. Zargar, A. Negrete, J.Shiloach, G.F. Payne, W.E. Bentley. Electronic control of gene expression and cell behaviour in Escherichia coli through redox signaling. Nat Commun, 8 (2017), p. 14030
  
 
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Latest revision as of 02:40, 22 October 2019


Bioelectric Relay Construct

This part will respond to the electric potential via various redox modulators and the transformed cell begins to have the activated transcription factor, which is SoxR protein. This transcription factor protein binds to the bidirectional promoter region, then cells translate more SoxR and LuxI.

Biology

The Relay part contains E.coli. intrinsic the bidirectional promoter region of the SoxRS regulon. The SoxR coding sequence is truncated and LuxI coding region is added followed by a ssRA tag. LuxI gene is originally from the bacterium A. fischeri and encodes Acyl-homoserine-lactose synthase. LuxI synthases Acyl-homoserine-lactose (AHL) which is known as autoinducing peptides. The peptides are released in the extracellular environment, and other cells are able to detect the change in concentration of this peptide. This autoinduction process is called quorum-sensing (QS).

Usage

In our device, we use multiple redox modulators to convert electric stimuli into chemical signals. [Fig 1] The toxin of Gram-negative bacterium Pseudomonas aeruginosa, Pyocyanin is used as the initiator of this redox cycle. Added pyocyanin is the oxidized form and it oxidizes SoxR Transcription Factor protein to activate. The ubiquitous Menaquinone transports an electron across the membrane and Pyocyanin is again oxidized. Ferrocyanide/Ferricyanide in the extracellular environment gets reduced via Menaquinone and the positive potential oxidizes Ferrocyanide to keep modulators oxidized. Therefore, the cycle keeps activating SoxR protein. The activated SoxR protein binds to the pSoxR/pSoxS bidirectional promoter region and expresses LuxI and SoxR proteins. SoxR protein is again activated under the positive electric potential as explained above. LuxI protein acts as Acyl-homoserine lactone (AHL) synthase. AHL will be diffused outside the cell and induces the surrounding cells' response to the change in the concentration of AHL.


Figure 1: Quorum Sensing by Electric Stimulation, this part is on the top

Characterization

NYU Shanghai iGEM team characterized this part by applying electric stimulus directly onto the agar plate that has necessary electromodulators and transformed cells with this Biopart. As we have known that 2.5uM Pyocyanin and 5mM Ferrocyanide(R)/5mM Ferricyanide (O) optimizes the activation of pSoxRS regulons, we mixed these amount of drugs in the given agar to be solidified in the plates with the two holes with electrodes. (Electrodes are covered by the agar so that cell culture is not exposed to the graphite.) Sodium sulfite is also added to create anaerobic environment on the plate.

On the other, BL21 is transformed with the pUC57 plasmid that contains this part, and inoculated overnight. 50 uL of fully inoculated BL21 and 350 uL of LB medium is mixed and spread on the prepared agar plate. 1 hour of the electric stimulus (0.5V) followed by culturing at 37˚C incubator overnight. The result is shown in Figures 2 and 3.

Figure 2: GFP Expression Level of Co-culture of Relay and Receiver part, Individual culture of Relay and Receiver, and No Cells. Every sample is stimulated by 0.5V potential for an hour.
Figure 3: Quorum Sensing by Electric Stimulation- UV

The quantification shows that this part enhances the GFP expression level when it is used with the Receiver cell (BBa_K3267001) and the role of Relay cells is showing that there are no colonies glowing around the positive electrodes (Left, Fig 3). These areas are for the receiver cell that responds to the positive electric potential while surrounding colonies are Receiver part cells (BBa_K3267001) which responded to the AHL gradient.

Reference

T. Tschirhart, E. Kim, R. McKay, H. Ueda, H.C. Wu, A.E. Pottash, A. Zargar, A. Negrete, J.Shiloach, G.F. Payne, W.E. Bentley. Electronic control of gene expression and cell behaviour in Escherichia coli through redox signaling. Nat Commun, 8 (2017), p. 14030

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1165
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 49