Revision as of 14:00, 30 October 2013

Variant of the wild-type pLuxR promoter with lower sensitivity

Figure 8: Sequence alignment of the library member luxR variants.

BBa_K1216008: part of a P_LuxR collection of promoters with differing AHL sensitivity. This significantly increases usefulness of the Lux signaling system, as differential respones to cell concentrations are enabled. Further they can even be used to detect geometrical settings of colonies.

After obtaining a less sensitive variant of P_LuxR we used it as a starting point for further engineering of mutant promoters. This approach ultimately lead to a library of promoters spanning a reasonably big range of EC₅₀ values. Please see details in the characterization section below or by clicking the characterization tab.

Usage and Biology

Figure 1: Overview of the Lux signaling system. With no LuxR-AHL complex bound, P_LuxL enables transcription of luxR whereas P_LuxR is shut off except for slight expression of the downstream operon. If AHL concentration rises inside the cell, P_LuxL is shut down and P_LuxR induced, yielding high expression of LuxI and thus further increase in AHL concentration, constituting positive feedback.

The Lux signaling system is based on positive feedback. The P_LuxL promoter drives expression of the upstream luxR gene. The promoter P_LuxR driver expression of the downstream operon including the LuxI AHL synthetase. The promoter encompasses a palindromic operator sequence to which a dimer of LuxR-AHL complexes binds to induce transcription. In absence of this complex, P_LuxL is active and thus LuxR is abundant. P_LuxR however is inactive, showing slight leakage, yielding a fairly low expression level of LuxI and other downstream gene products.

An important feature of AHL, the "signaling molecule" of the system, is that it diffuses freely through cell walls and surrounding media. This enables communication between cells in vincinity. When a certain cell concentration is reached, the diffusing AHL yields sufficiently high levels of the LuxR-AHL complex within cells. The direct consequence of this is high expression of downstream genes, importantly inculding LuxI (and not so importantly shutting down P_LuxL). Now the positive feedback loop is being closed by elevated synthesis of AHL which further increases LuxI synthesis in cells.

In V. Fischeri other downstream genes include a luciferase which is part of a symbiosis with marine animals. Other possibilities are proteins that facilitate biofilm formation and related phenomena.

In (synthetic) biology, this system can be used as a sender/reporter system by separating LuxI and LuxR, where LuxI is not under control of P_LuxR, rendering the system independent of cell concentration. LuxR/P_LuxR can then be used to drive expression of a myriad of other genes such as various reporter genes.

Sequence and Features

Characterization

Figure 9.Dose response curve experiment plates of the promoter library with an RFP reporter. ON the picture you can see 11 plates having different AHL concentrations in the agar( 10^-5 nM, 10^-4 nM, 10^-3 nM,10^-2 nM,10^-1 nM,10^0 nM,10^1 nM,10^2 nM,10^3 nM,10^4 nM,10^5 nM.) of the backmutations SM1, SM2, SM3, SM3, S, SM5, SM6 and DM. (SM = Single mutation, DM = Double mutation.

As explained above we created a library of promoters originating from P_LuxR variant (G1). We characterized the dose response curve to AHL on agar plates of all 7 promoters by following the same protocol as for the P_LuxR variant characterization. See the different shifted dose response curves in Figure 10. and the characteristics of the library in Table 1.

Thanks to the analytical solution of the model (see [http://2013.igem.org/Team:ETH_Zurich/Modeling/Analytical_Approximations here]) predicting the needed EC₅₀ of 100 nM of the promoter to fit to our system we choose the SM1 mutant for our final set-up. (We did not choose SM2 because when we choose a promoter, according to the prediction of the model, the experiments were not done in duplicates at this time, and the characteristics changed of course as soon as we had duplicates). This promoter gets activated when two mines are nearby the receiver cell.

Table 1. Characteristics of the promoter library

Figure 10. Dose response curve of the promoter library. 7 different promoter were created by partial sensitivity recovery of the P_LuxR variant. The colonies have the P_Lac-LuxR-P_LuxR BBa_J09855 construct with the different mutated promoter as well as a RFP reporter to analyze the dose response in the flow cytometer. The colonies were grown on 11 different plates with 11 different concentrtions as explained in Figure 9. Please see table 1 for the characteristics of the promoter library.

Figure 11. Comparison of a choosen set of promoters. The wild-type, P_LuxR variant (G1, and the mutants created by rational design and partial sensitivity recovery SM1 and SM5.

Figure 10. shows the relative fluorescence over the [AHL] of the dose-response curves of the whole library exepted the G1 and the wild type P_LuxR promoter. Figure 11. is a comparison of a choosen set of promissing promoter we tried in different constructs for the final gameplay according to the prediction of the model.

Fluorescence data analysis

The fitting of the following graphs was performed using this equation :

Output = eGFP levels [au]
Top = maximal eGFP level [au] ("full induction")
Bottom = minimal eGFP level [au] (“leakiness”)
n = Hill coefficient (“cooperativity”)
EC₅₀ = Half-maximal effective concentration (“sensitivity”)
[AHL]=AHL concentration [nM]

References