Difference between revisions of "Part:BBa K1216007"

Revision as of 18:23, 4 October 2013

Variant of the wild-type pLuxR promoter with lower sensitivity

BBa_K1216007 is a variant of the wild-type pLuxR promoter with a lowered sensitivity for LuxR-AHL.

Comparison of BBa_R0062 to the mutant

Sequence and Features

Characterization

Site saturation directed mutagenesis of the BBa_R0062 pluxR wild type promoter to obtain the pluxR variant BBa_K2126007

Figure 1.Location of the site directed saturation mutagenesis in the luxbox.As indicated you can see the mutations of the pluxR variant compared to the wild type in red. The positions which were mutated randomly are indicated in red-underlined

Our goal was to achieve mutations in the luxbox of the pLuxR BBa_R0062 promoter to shift the dose response curve of the promoter depending on the OHHL (3-oxo-hexanoyl-l-homoserine-l-lactone) concentration.
We did site directed saturation mutagenisis of specific sites of the luxbox according to literature ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2446796/ Luis Caetano A Mutational Analysis Defines Vibrio fischeri LuxR Binding Sites])
We mutated the promoter directly in the BBa_J09855 construct and cloned a GFP gene as reporter to be able to screen for different dose response curves. In the end we isolate one mutated promoter which shows a shift in sensitivity. For more details see : Characterization of the pLuxR variant dose response to OHHL using plate reader analysis and FACS single cell analysis

Characterization of the pLuxR variant dose response to OHHL using plate reader analysis and FACS single cell analysis

Characterization and comparison to the wild type pLuxR using the plate reader analysis

Figure 1. Sensivity curve of the wild type promoter and pLuxR variant (G1) analyzed on the Tecan M200 plate reader. The OHHL 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.

The GFP reporter allows us to make fluorescence measurement in plate reader experiments as well as in single cell analysis to characterize the promoter behavior depending on [OHHL], by recording the fluorescence [au]. According to literature [http://www.ncbi.nlm.nih.gov/pubmed/18760602 (Geske G.D. Evaluation of a focused library of N-aryl L-homoserine lactones reveals a new set of potent quorum sensing modulators)] we could define the first experimental OHHL ranges to induce the promoter. After some fine tunning and adjusting experiment we could narrow down the ranges to obtain a dose response curve in a reasonable resolution for the BBa_R0062 wild type promoter to the OHHL (3-oxo-hexanoyl-l-homoserine-lactone) concentration. See Figure 1. This datas were obtained from plate reader experiment. Accordnig to the sensivity range of the wild type promoter we could screen for promoters, and scan the potentially shifted variants in different ranges of [OHHL]. We finally isolate one promoter variant which shows a shift compared to the wild type. See G1 on Figure 1. The pLuxR variant has an EC₅₀=1'341 nM , the pLuxR wild type has an EC₅₀=5.86 nM according to the plate reader data. The single cell analysis gave us data of higher quality (see below).

Characterization of the pLuxR dose response to OHHL in liquid culture and on agar plates by FACS single cell analysis

To obtain high quality data we did single cell analysis over the dose response range defined above. Obviously the EC₅₀ change compared to the plate reader data. Figure 2.

Figure 2.Single cell data of the wild type BBa_R0062 promoter vs. the pluxR mutant. The histogram plots of eGFP levels for each tested OHHL 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.

Figure 3. Dose response curve of the pLuxR variant G1 on agar plates and liquid culture. The black curve shows the response of to OHHL in liquid culture, the orange curve shows the response to OHHL on agar plates
For the liquid culture we got :EC₅₀=6'482nM, R²=0.97, n=0.8;
For the agar plates we got :EC₅₀=17'814nM, R²=0.93, n=0.8

The plot shows the dose reponse curve of the promoter depending on OHHL concentration. We performed this experiment in liquid culture as well as on agar plates (OHHL was added to the melted 1.5% agar), which was closer to our project. We observed a shift in the dose response between the liquid culture (EC₅₀=6'482nM) single cell analysis of and the one on agar plates(EC₅₀=17'814nM). See Figure 3

Comparison of the pLuxR variant and pLuxR Wild-Type dose response to OHHL in liquid culture and on agar plates

Figure 4. Comparison of the sensivity of the wild type promoter and the mutated PluxR1 promoter on agar plates
For the wild type we got :EC₅₀=4.45nM, R²=0.80, n=1.7
For the pLuxR variant we got :EC₅₀=17'814nM, R²=0.93, n=0.8

The plot shows the comparison between the wild type pLuxR and pLuxR variant dose reponse curve to [OHHL] on agar plates (OHHL was added to the melted 1.5% agar), which was closer to our project. The shift in the dose response between the wild type pLuxR (EC₅₀=4.45 nM and the pLuxR variant(EC₅₀=17'814nM) is about 4003 times less sensitive according to the single cell analysis. See Figure 4

Figure 5. Comparison of the sensivity of the wild type promoter and the mutated PluxR1 promoter in liquid culture
For the awild type we got :EC₅₀=0.02 nM, R²=0.84, n=1.7
For the pLuxR variant we got :EC₅₀=6'462nM, R²=0.97, n=0.8;

The plot shows the comparison between the wild type pLuxR and pLuxR variant dose reponse curve to [OHHL] in liquid cultures. The shift in the dose response between the wild type pLuxR (EC₅₀=0.02 nM and the pLuxR variant(EC₅₀=6'462 nM) is about 300'000 times different according to the single cell analysis. See Figure 4