Positive and negative selection of the cells transformed with mutated P_LuxR</br>

Figure 1: Two sample plates used in the selection process. a Plate containing AHL (100 nM). All bright colonies have either a promoter that is sensitive to AHL or a constitutive one. b Plate without AHL. All bright colonies have a constitutive promoter and can be ruled out.

At first we incubated the transformed cells on an AHL containing plate to rule out all AHL-insensitive mutations. All colonies expressing GFP were then restreaked on a plate which contained no AHL, thus allowing us to rule out constitutive promoters. The remaining clones were then grown in luquid cultures on 96-well plates and tested for sensitivity over a broad range of AHL concentrations. Promising candidates were then measured in a flow cytometer to check for bistable promoters.

Figure 2: Sensivity curve of the wild type promoter and G1 mutant. The AHL concentration range is plotted against the standarized fluorescence (au).
For the wild type: EC₅₀=5.86 nM, R₂= 0.87,n=1.7
Fore the G1 mutant: EC₅₀=1'341 nM, R₂= 0.98,n=0.8. All assays were carried out in triplicates, results are presented as mean ± standard deviation.

Preliminary tests on microtiter plate reader screening

First of all we did a fast and preliminary screening of the mutated PluxR promoters with the microtiter plate reader. Thus, we carried out dose response curves using the Tecan Infinite M200 plate reader. The test range was inspired from literature (1). Afterwards we did flow cytometry of the most promissing promoters to have high quality data and exclude bistable promoters.

The results are shown in Figure 2.

Dose response curve single cell analysis of the wild-type P_LuxR and the first P_LuxR variant(G1) Liquid culture and agar plate AHL detection comparison

Here we employed single cell flow cytometry to obtain high quality fluorescence data of each promoter under different AHL concentrations.we started by analyzing cells grown in liquid culture, then we shifted to agar plate format in order to fit to our project. We obtained different EC₅₀ values between cells in liquid culture and in agar plates for each luxR promoter (see Figure 3, 4 and 5). See methods for the protocol

Figure 3: Sensitivity curve of the wild type BBa_R0062 on agar plates and liquid culture For the liquid culture we got :EC₅₀=0.02 nM nM, R²=0.84, n=1.7; For the agar plates we got :EC₅₀=4.45 nM, R²=0.80, n=1.7. All assays were carried out in duplicates, results are presented as mean ± standard deviation.

Figure 4: Single cell data of the wild type BBa_R0062 promoter vs. the pluxR mutant The histogram plots of eGFP levels for each tested AHL concentration, culturing condition and corresponding promoter. In orange experiments on agar plates. In black experiments in liquid cultures. On the top the data of the wild type luxR promoter. On the bottom the data of the luxR promoter variant. All assays were carried out in duplicates, results are presented as mean ± standard deviation.

Figure 5: Sensitivity curve of the G1 (pluxR variant BBa_K1216007) on agar plates and liquid culture
For the liquid culture we got: EC₅₀=6482 nM, R²=0.97, n=0.8;
For the agar plates we got :EC₅₀=12'555nM, R²=0.93, n=0.8. All assays were carried out in duplicates, results are presented as mean ± standard deviation.

In liquid culture

The direct plot of both sensitivity curves (Figure 6) shows the shifted sensitivity of the luxR promoter in comparison to the wild type BBa_R0062 promoter. The EC₅₀ shifted from 0.02nM for the wild type BBA_R0062 to 6'250 nM for the luxR variant promoter which is equal to a 300'000 fold increase.

Figure 6: Comparison of the Wild Type pluxR and the pluxR mutant sensitivity curves in liquid culture using the BD LSRFortessa™ Flow Cytometer System
For the wild type :EC₅₀=0.02nM, R²=0.84, n=1.7
For G1 we got :EC₅₀=6482nM, R²=0.97, n=0.8. All assays were carried out in duplicates, results are presented as mean ± standard deviation.

On agar plates

The direct plot of both sensitivity curves (Figure 6) shows the shifted sensitivity of the P_luxR promoter in comparison to the wild type BBa_R0062 promoter. The EC₅₀ shifted from 4.45 nM for the wild type BBA_R0062 to 12'555 nM for the luxR variant promoter which is equal to a 2800 fold increase. Therefore, comparing with the results from cells growing in liquid culture an increase of 220 and 2 times for wild type BBa_R0062 and pluxR variant respectively. If you want to know more about the methods please click here

Figure 7: Comparison of the sensivity of the wild type promoter and the mutated luxR promoter on agar plates
For the wild type we got :EC₅₀=4.45 nM, R2=0.80, n=1.7
For the pLuxR variant we got :EC₅₀=12'555 nM, R2=0.93, n=0.8. All assays were carried out in duplicates, results are presented as mean ± standard deviation.

Rational design and single cell analysis of additional P_LuxR variants with partial sensitivity recovery based on the first P_LuxR variant (G1)

Figure 8: Sequence alignment of the additional luxR variants.

According to initial model predictions confirmed by successive experimental validation the G1 promoter sensitivity is too low to drive a significant response in the concentration gradient established by our sender cells.
We need a collection of promoters with a set of EC₅₀ values between the wild type and G1 itself. Since G1 has been obtained through two random mutations: 4T>A and 16C>G reverting one of the two, we reasoned, should result in a LuxR binding strength closer to the wild type. To test this hypothesis we ordered oligos encompassing all the possible combination of single position mutants (See list) and plan to experimentally characterize, and eventually deposit in the registry, all the resulting pLux variants. In parallel we used degenerate oligos to generate a small library (16 members) containing all the combination of double mutants for the two G1 key position to verify if all the double mutants show similar EC₅₀.

Finally characterized promoter library

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.

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 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.

Table 1. Characteristics of the promoter library