Difference between revisions of "Part:BBa K2972008"

Latest revision as of 03:50, 22 October 2019

Quorum sensing(QS) sensitivity test

Introduction

This part is used to test the sensitivity of QS system and this part is improved from the previous BBa_K1499500 so that it will be more suitable for our project and can be used more widely.
To discover the relationship between the components produced by the QS system and the density of cells in the process of cell growth. We obtained the curve of fluorescence intensity changing with time under different promoters which inducing LuxR expression according to experiments.

Modeling

When considering the change of signal factor concentration in a single cell, we refer to the model of Maike in 2010. Because our model is in a closed container, we delete the diffusion term from the original model, and improve the solvability. We modified and established our own model according to the actual situation.

Result

In this result, we can clearly see that AHL showed very low growth rate in the earlystage. At a certain time, when the population density reached a certain threshold,AHL showed a sharp increase trend, indicating that quorum sensing has occurred. The growth rate of LuxI also began to increase suddenly, and a large number of products were produced. This result shows that our model is very consistent with the phenomenon of quorum sensing, and can be used as a mathematical description of the cell growth state in the experiment. This model can be great significance to the experimental guidance.

Experiment

QS system verification experiment
The production of seed fermentation broth：The sequenced strains were transferred to petri dishes and activated（The strain information is shown in table 1），Then we transferred the activated strain to a single colony in LB medium containing 0.1% Amp. Shake flask fermentation ：

Add 20ml LB medium containing 0.1%Amp into 50ml conical flask
Measure seed fermentation broth concentration with an ultraviolet spectrophotometer
Transfer the seed fermentation broth to 20mlLB medium（0.1%Amp），when the OD600.is0.05Abs
Culture in shaking table for 12h, sampling every 2 hours

Fluorescence values and OD600 data at the excitation wavelength of 480nm and emission wavelength of 517nm were measured and the results were as follows：

Fig1.the curve of fluorescence intensity changing with time under different promoters

Result analysis

Analysis of the results: Since the strain transferred from the seed solution to the Erlenmeyer flask has opened the QS system, green fluorescence has been expressed, so we used the initial fluorescence value after four hours of culture. From Fig. 1, we can see that as the fermentation time progresses, the green fluorescence value gradually increases, and the strain QS system is turned on. In the promoter of choice, the J23102 promoter is insensitive to quorum sensing signals, resulting in unstable expression of green fluorescent protein. The experimental results show that the J23117 promoter is more suitable for use in E. coli.

Gold medal part improvement

Through the promoter strength instructions provided by the IGEM official website, we performed different intensity promoter replacement experiments on the QS population sensing line. The effect of different LuxR content on the QS system and the growth of the bacteria itself was realized. The experimental results showed that the promoter J23102 with high intensity was not sensitive to the population density of E. coli. Although J23102 can open the QS population density sensing line, the promoter J23117 is more suitable for the opening of the QS population density system in E. coli.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 2359
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1634

@@ Line 4: / Line 4: @@
 ===Introduction===
-This part is used to test the sensitivity of QS system.<br>
+This part is used to test the sensitivity of QS system and this part is improved from the previous BBa_K1499500 so that it will be more suitable for our project and can be used more widely.<br>
 To discover the relationship between the components produced by the QS system and the density of cells in the process of cell growth. We obtained the curve of fluorescence intensity changing with time under different promoters which inducing LuxR expression according to experiments.
@@ Line 11: / Line 11: @@
 When considering the change of signal factor concentration in a single cell, we refer to the model of Maike in 2010. Because our model is in a closed container, we delete the diffusion term from the original model, and improve the solvability. We modified and established our own model according to the actual situation.
+[[File:BIT-China gold2.png|600px]]
 [[image:BIT-China Modeling3.jpg|400px]]
 [[image:BIT-China Modeling4.jpg|400px]]
@@ Line 29: / Line 29: @@
 Fluorescence values and OD600 data at the excitation wavelength of 480nm and emission wavelength of 517nm were measured and the results were as follows：
-[[File:BIT-China gold1.png|500px|thumb|center|<b>Fig1.the curve of fluorescence intensity changing with time under different promoters<\b>]]<br>
+[[File:BIT-China gold1.png|500px|thumb|center|<b>Fig1.the curve of fluorescence intensity changing with time under different promoters</b>]]<br>
 ====Result analysis====
-Because the strain transferred from the seed fermentation broth to the conical flask has turned on the QS system and the green fluorescence has been expressed, we took the initial fluorescence value after culture for four hours.As can be seen from figure 1, with the passage of fermentation time, the green fluorescence value gradually increased, and the QS system of the strain was turned on. However, after two hours of opening, excessive quorum sensing signals inhibited the pLUX promoter, leading to the gradual shutdown of the QS system.The low expression of the strong promoter J23102 in the QS strain of green fluorescent protein indirectly indicates that quorum sensing signal molecules have negative effects on the QS system. With the decrease of quorum sensing semaphore, pLUX can be turned on normally to realize the expression of green fluorescent protein. After 10 hours of culture, fluorescence was no longer significantly increased or decreased. Maybe the nutrient content in the culture medium was too low, which affected the growth of escherichia coli, and the population density would not increase too much.The 12h growth curve is shown in figure 2.
+Analysis of the results: Since the strain transferred from the seed solution to the Erlenmeyer flask has opened the QS system, green fluorescence has been expressed, so we used the initial fluorescence value after four hours of culture. From Fig. 1, we can see that as the fermentation time progresses, the green fluorescence value gradually increases, and the strain QS system is turned on. In the promoter of choice, the J23102 promoter is insensitive to quorum sensing signals, resulting in unstable expression of green fluorescent protein. The experimental results show that the J23117 promoter is more suitable for use in E. coli.
 ====Gold medal part improvement====
-According to the description of promoter strength provided by the official website of IGEM, we conducted different strength promoter replacement experiments for QS quorum sensing circuit.We investigated the effects of different LuxR contents on QS system and the growth of bacteria.The results showed that promoter J23102 with high intensity was not sensitive to the population density of e. coli..Although J23102 can realize the opening of QS population density induction circuit, the promoter J23117 with the weakest expression intensity is more suitable for the opening of QS population density system in e. coli.At the same time, the change of different promoters indicated that LuxR would not affect the growth of e. coli to some extent.
+Through the promoter strength instructions provided by the IGEM official website, we performed different intensity promoter replacement experiments on the QS population sensing line. The effect of different LuxR content on the QS system and the growth of the bacteria itself was realized. The experimental results showed that the promoter J23102 with high intensity was not sensitive to the population density of E. coli. Although J23102 can open the QS population density sensing line, the promoter J23117 is more suitable for the opening of the QS population density system in E. coli.