Difference between revisions of "Part:BBa K3267001"

Latest revision as of 02:43, 22 October 2019

Quorum-Sensing Receiver Construct

Transformed cells with this construct respond to AHL-dependent quorum sensing and GFP would be expressed by the activated transcription factor protein LuxR which binds to the bidirectional promoter region (pLuxR/pLuxI).

Biology and Usage

This Receiver part contains the V. fischeri-intrinsic bidirectional promoter region, the LuxRS regulon. The high concentration of Acyl-homoserine lactose (AHL) binds to the LuxR protein to activate and it binds to pLuxR/pLuxS promoters. Instead of LuxS protein, we add super folder GFP in order to make this part as the reporter part of the whole system (Fig 1). The super folder GFP is followed by the PixCell degradation tag from the 2018 Imperial College team to analyze the reporter gene spatiotemporally.

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

Characterization

Bioelectric Relay cell and Receiver cell (BBa_K3267001 and this part) are separately transformed in E. coli. (BL21 strain) and mixed on the agar plate to cultured under electric stimulation. The agar plate is made with necessary electrochemical modulators: 2.5 uM Pyocyanin; 5mM Ferricyanide; 5mM Ferrocyanide; and 0.02% Sodium sulfite. The GFP fluorescence level is determined by pixel intensities after a 1-hour electric shock followed by 24-hour post-culturing. There are three sets to show our model: Co-culture of both cells, only relay cell, and only receiver cell.

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: Co-culture Plate under GFP (the left hole is positive anode; the right hole is the negative cathode)

Quantitatively, only when both Relay and Receiver cell exists (Co-culture), GFP expression is enhanced. Only the Receiver cell has lightly more expression of GFP than only Relay cell (Fig 2). This should be due to the intrinsic LuxR proteins that facilitate the GFP expression only by its own plasmid. Qualitatively, we also observed the spatial expressing patterns of quorum sensing as most of the GFP expressing cells are located as if the glowing colonies are surrounding the positive electrode on the left side (Fig 3). This is because AHL molecules diffused from the Relay cells around the positive electrode and Receiver cells responded to the gradient of Quorum-sensing inducers.

Improvement Upon BBa_K2862021

We made this electrochemical control of Bio-parts possible with simpler equipment and a more complicated pathway. 2018 Imperial College team used a commercial potentiostat, but we handmade the electrodes connected to the simple power supply in the physics lab, which significantly saves our budget and time. The electric modulation worked properly in a more complicated setting as we discussed above. The existing part only used SoxRS regulons with the reporter gene, but our system extended the research on electric control of bacterial genes with LuxRI regulons with two layers signaling.

Figure 4: Electric Modulation On Agar Plate Setup

Figure 5: Making Sure that the Electric Setup has 0.5 V

Referece

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