Team:ETH Zurich/lab/biobrick/used1


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<partinfo>BBa_R0062 AddReview 4</partinfo> ETH Zurich 2014


Characterization of the promoter's basal leakiness to LuxR in the absence of 3OC6-HSL

The amount of regulator LuxR (BBa_C0062) in the system was shown to influence the pLuxR promoter's basal expression or leakiness. By using the three different constitutive promoters BBa_J23100, BBa_J23109, and BBa_J23111 for the production of LuxR we have measured this effect in terms of fluorescence.

Background information

We used an E. coli TOP10 strain transformed with two medium copy plasmids (about 15 to 20 copies per plasmid and cell). The first plasmid contained the commonly used p15A origin of replication, a kanamycin resistance gene, and promoter pLuxR (BBa_R0062) followed by RBS (BBa_B0034) and superfolder green fluorescent protein (sfGFP). In general, for spacer and terminator sequences the parts BBa_B0040 and BBa_B0015 were used, respectively. The second plasmid contained the pBR322 origin (pMB1), which yields a stable two-plasmid system together with p15A, an ampicillin resistance gene, and one of three promoters chosen from the Anderson promoter collection followed by luxR (BBa_C0062). The detailed regulator construct design and full sequences (piG0041, piG0046, piG0047) are available here.

Experimental Set-Up

The above described E. coli TOP10 strain was grown overnight in Lysogeny Broth (LB) containing kanamycin (50 μg/mL) and ampicillin (200 μg/mL) to an OD600 of about 1.5 (37 degrees Celsius, 220 rpm). As a reference, a preculture of the same strain lacking the sfGFP gene was included for each assay. The cultures were then diluted 1:40 in fresh LB containing the appropriate antibiotics and measured in microtiter plate format on 96-well plates (200 μL culture volume) for 10 h at 37 degrees Celsius with a Tecan infinite M200 PRO plate reader (optical density measured at 600 nm; fluorescence with an excitation wavelength of 488 nm and an emission wavelength of 530 nm).

Modeling leakiness


Table 1 Crosstalk matrix for the promoter pLux (BBa_R0062)

In all the measurements conducted to create this matrix the promoter pLux was the basis and was induced in six different variations shown. The dark blue points in the graph top left show the activation of gene expression when pLux is induced by 3OC6-HSL (Lux-AHL) binding to the corresponding LuxR regulator. The observed transition occurs at a concentration of approximately 1 nM of 3OC6-HSL. The light-blue curve plotted shows modeling data of pLux induced by 3OC6-HSL (Lux-AHL) binding to the corresponding LuxR regulator. This curve from the model and the dark blue data points obtained from experiments were plotted as a reference in all the other graphs describing pLux. Crosstalk can be observed for the cases where the 3OC12-HSL (Las-AHL) binds the LuxR regulator. Additionally for 3OC12-HSL binding to its corresponding regulator LasR and then binding to the pLux as seen in the middle of the top row and center of the matrix. For the case of Las-AHL binding the regulator LasR and subsequently the promoter pLux, the transition occurs at 1 nM and reaches 0.5 fold the fluorescence as pLux induced by 3OC6-HSL binding LuxR. In the case of 3OC12-HSL binding LuxR and inducing the promoter pLux, the transition is observed at approximately 100 nM and severe crosstalk is observed, meaning that the ON-OFF-ratio is not significantly different from the reference curve.

Observation of C4-HSL has shown, that there is no significant crosstalk with the LuxR regulator and LasR regulator binding C4-HSL and subsequently to pLux. This is indicated on top right and middle right graphs. However, RhlR induced with its corresponding inducer (C4-HSL) binds to pLux and activates expression of GFP at about 100 nM.

ETH Zurich 2014 qs-table CornerLux.png ETH Zurich 2014 qs-table 3OC6-HSL.png ETH Zurich 2014 qs-table 3OC12-HSL.png ETH Zurich 2014 qs-table C4-HSL.png
ETH Zurich 2014 qs-table LuxR.png ETH Zurich 2014 qs-table PluxRef.png ETH Zurich 2014 qs-table PluxLuxRLasAHL.png ETH Zurich 2014 qs-table PluxLuxRRhlAHL.png
ETH Zurich 2014 qs-table LasR.png ETH Zurich 2014 qs-table PluxLasRLuxAHL.png ETH Zurich 2014 qs-table PluxLasRLasAHL.png ETH Zurich 2014 qs-table PluxLasRRhlAHL.png
ETH Zurich 2014 qs-table RhlR.png ETH Zurich 2014 qs-table PluxRhlRLuxAHL.png ETH Zurich 2014 qs-table PluxRhlRLasAHL.png ETH Zurich 2014 qs-table PluxRhlRRhlAHL.png

Characterization of the promoter's sensitivity to 3OC6-HSL depending on LuxR concentration

Background information

Systems considered

Modeling promoter's sensitivity


Characterization of two-level crosstalk on the promoter

Background information

System considered

Modeling crosstalk

First-order crosstalk

First Level crosstalk: LuxR binds to different HSL and activates the promoter

ETH Zurich 1crosstalkPlux.png

Second Level crosstalk: other regulatory proteins, like LasR, bind to their natural HSL substrate and activates the promoter

ETH Zurich 2crosstalkPlux.png

Second order crosstalk: Combination of both cross-talk levels

Other regulatory proteins, like LasR, bind to different HSL and activates the promoter

ETH Zurich 3crosstalkPlux.png