In this study, a simple and easy-to-use engineering microbial ' safety lock ' was designed at 37 °C ( the temperature in the human body, that is, the working temperature of probiotics; it is also a common temperature for the fermentation of some industrial microorganisms ). Under this condition, the ' safety lock ' will not work, and the host engineering bacteria can reproduce and work normally. At 22 ° C ( closer to the temperature in the natural environment ( except in tropical regions and hot summers ) ), the “safety lock ” plays a role in expressing a protein that is toxic to the host engineering bacteria, causing the host to “commit suicide” , thereby preventing the leakage of the engineering bacteria. To verify whether the temperature control system is regulated by temperature and the influence of different temperatures on the temperature control system ( pTRIP-EGFP ), and to study whether the temperature control system is open or closed when the temperature is lower than 22 degrees; when the temperature is 22-37 degrees, the temperature control system is in the open state or closed state; to further find the most suitable temperature for EGFP protein expression. In addition, we tested the function of the temperature-controlled switch pTRIP-ccdB in E.coli DH5α. We wanted to find out the optimal induction concentration and time of pTRIP-ccdB growth ability (OD600) by modeling and adjust the temperature control for adjustment.

1. Date

1.1 The fluorescence intensity of EGFP in pTRIP-EGFP

We set different temperatures, the independent variables were 16^oC,19^oC,22^oC,26^oC，32^oC，and37^oC degrees, the dependent variable was the fluorescence intensity of EGFP. The data showed that the fluorescence intensity of EGFP increased first and then decreased with the increase of temperature ( Table 1 ). There may be a maximum value for the fluorescence intensity of EGFP between 19^oC and 26^oC, so we use modeling to predict the temperature at which the fluorescence intensity of EGFP is the largest.

Table 1.Fluorescence intensity of EGFP at different temperatures

 

1.2 pTRIP-ccdB OD600 growth ability

In order to find the effect of ccdB on E.coli DH5α, we set different concentrations of N- ( 3-oxocaproyl ) -L-homoserine lactone ( AI ) and different time of pTRIP-ccdB strain growth ability ( OD600 ). we set different concentrations of N- ( 3-oxocaproyl ) -L-homoserine lactone ( AI ) and different time of pTRIP-ccdB strain growth ability ( OD600 ) ; the independent variables were time and N- ( 3-oxohexanoyl ) -L-homoserine lactone ( AI ), and the dependent variable was OD600. The time was 2.5h, 5h, 10h, 20h, 30h ; the concentrations of N- ( 3-oxohexanoyl ) -L-homoserine lactone ( AI ) were 0.2 mmol, 0.4 mmol, 0.6 mmol, 0.8 mmol and 1 mmol ( Table 2 and Table 3) ;

 

Table 2. OD600 of pTRIP-ccdB at 22 °C

AI(mmol) /Time（h）	0.20			0.40			0.60			0.80			1.00
2.5	0.19	0.18	0.19	0.18	0.16	0.15	0.11	0.13	0.13	0.10	0.09	0.11	0.11	0.1	0.11
5.O	0.20	0.21	0.22	0.19	0.19	0.20	0.16	0.13	0.15	0.16	0.14	0.13	0.13	0.15	0.14
10	0.24	0.15	0.26	0.24	0.25	0.26	0.18	0.18	0.16	0.20	0.21	0.23	0.22	0.19	0.23
20	0.27	0.25	0.29	0.26	0.24	0.26	0.19	0.16	0.2	0.25	0.24	0.26	0.26	0.25	0.26
30	0.31	0.30	0.29	0.26	0.27	0.25	0.21	0.22	0.2	0.26	0.27	0.25	0.26	0.25	0.26

 

Table 3. OD600 of pTRIP-ccdB at 37 °C

AI(mmol) /Time（h）	0.20			0.40			0.60			0.80			1.00
2.5	0.34	0.35	0.32	0.33	0.31	0.32	0.37	0.35	0.38	0.29	0.3	0.31	0.29	0.32	0.31
5.0	0.81	0.79	0.82	0.89	0.82	0.85	0.87	0.85	0.83	0.81	0.82	0.8	0.8	0.79	0.81
10	1.03	1.09	1.00	0.99	1.06	1.14	1.14	1.21	1.18	1.09	1.05	1.1	1.02	0.99	0.99
20	1.21	1.24	1.23	1.2	1.21	1.25	1.2	1.25	1.27	1.23	1.19	1.15	1.29	1.28	1.26
30	1.36	1.35	1.33	1.32	1.34	1.35	1.31	1.34	1.38	1.30	1.29	1.31	1.28	1.29	1.28

 

 

2. Modeling process and results

2.1 The fluorescence intensity of EGFP of pTRIP-EGFP

According to data analysis, it is a very typical data with Gaussian characteristics, which is large in the middle and small on both sides. Therefore, the Gaussian function model is established, and the coefficients of the function are calculated by using the nonlinear fitting toolbox of Matlab.

The Gaussian model process modeling process is as follows :

The code is as follows :

%% Fit: 'untitled fit 1'.

clear;clc

x0=[16 19 22 26 32 37];

y0=[4.45e+03 1.82e+04 3.99e+04 2.11e+04 3.59e+03 8.46e+02];

%% Fit: 'untitled fit 1'.

[xData, yData] = prepareCurveData( x0, y0 );

 

% Set up fittype and options.

ft = fittype( 'gauss2' );

opts = fitoptions( 'Method', 'NonlinearLeastSquares' );

opts.Display = 'Off';

opts.Lower = [-Inf -Inf 0 -Inf -Inf 0];

opts.StartPoint = [39900 22 1.32668631662499 21095.5016018701 26 3.00906644765801];

 

% Fit model to data.

[fitresult, gof] = fit( xData, yData, ft, opts );

 

% Plot fit with data.

figure( 'Name', 'untitled fit 1' );

h = plot( fitresult, xData, yData );

legend( h, 'y0 vs. x0', 'untitled fit 1', 'Location', 'NorthEast' );

% Label axes

xlabel x0

ylabel y0

grid on

The prediction model is Gaussian model :.The fitted parameter values refer to the following figure(Figure 1).

Figure 1. The fitted parameter values.

 

 

Modeling results :

According to Figure 2, it can be seen that the fluorescence intensity of EGFP increases first and then decreases with the increase of temperature. The maximum value appears at the temperature of 22.66, and the EGFP is 4.089e + 04, which proves that the fluorescence intensity of EGFP is the largest at 22.66 degrees. At the same time, it can be seen that in 16-22.66, EGFP showed an upward trend, indicating that the 16-22 degree temperature control switch gradually opened with the temperature ; when the temperature exceeds 22.66-37 degrees, the temperature control system is gradually closed. Therefore, it can be proved that EGFP will be expressed in large quantities at 22 degrees for a long time, which proves the feasibility of the temperature control system.

 

Figure 2. Model of EGFP fluorescence intensity at different temperature

 

2.2 OD600 growth ability of pTRIP-ccdB

2.2.1 OD600 of pTRIP-ccdB at 22 °C

The modeling code is as follows :

clear;clc

x0=[0.2 0.4 0.6 0.8 1];

y0=[2.5 5 10 20 30];

z0=[0.19 0.16 0.123333333 0.10 0.106666667;

0.21 0.19 0.146666667 0.14 0.14;

0.22 0.25 0.173333333 0.21 0.213333333;

0.27 0.25 0.183333333 0.25 0.256666667;

0.30 0.26 0.21 0.26 0.256666667];

x=[0.2:0.01:1];

y=[2.5:0.5:30];

[xi,yi]=meshgrid(x,y);

z=interp2(x0,y0,z0,xi,yi,'spline');

surfc(xi,yi,z)

[i1,j1]=find(z==min(min(z)));

[i2,j2]=find(z==max(max(z)));

xmin=x(j1)

ymin=y(i1)

zmin=min(min(z))

xmax=x(j2)

ymax=y(i2)

zmax=max(max(z))

The two-dimensional relationship between OD600 and time and concentration was obtained, and the response surface was made. The image is as follows :

Figure 3. The response surface of OD600 of pTRIP-ccdB with time and concentration at 22^o

 

According to the model calculation, the minimum OD600 was 0.0981, the corresponding concentration was 0.87, and the time was 2.5 h. It was speculated that the toxic protein ccdB began to express at about 2.5 h. It was predicted that the concentration of AI was 0.87 at 22 degrees, which was suitable for the expression of pTRIP-ccdB. Subsequently, some data of 0.5-0.8 mmol could be supplemented for fitting verification.

2.2.2The OD600 of pTRIP-ccdB at 37^o

The modeling code is as follows :

clear;clc

x0=[0.2 0.4 0.6 0.8 1];

y0=[2.5 5 10 20 30];

z0=[0.34 0.32 0.366666667 0.3 0.306666667;

0.81 0.853333333 0.85 0.81 0.8;

1.04 1.063333333 1.176666667 1.08 1;

1.23 1.22 1.24 1.19 1.276666667;

1.35 1.336666667 1.343333333 1.3 1.283333333];

x=[0.2:0.01:1];

y=[2.5:0.5:30];

[xi,yi]=meshgrid(0.2:0.01:1,2.5:0.5:30);

z=interp2(x0,y0,z0,xi,yi,'spline');

surfc(xi,yi,z)

[i1,j1]=find(z==max(max(z)));

[i2,j2]=find(z==min(min(z)));

xmax=x(j1)

ymax=y(i1)

zmax=max(max(z))

xmin=x(j2)

ymin=y(i2)

zmin=min(min(z))

The two-dimensional relationship between OD600 and time and concentration was obtained, and the response surface was made. The image is as follows :

Figure 4. The response surface of OD600 of pTRIP-ccdB with time and concentration at 37^o

 

According to the model(Figure 4), the maximum OD600 was calculated to be 1.3762, the corresponding concentration was 1mmol, and the time was 25.5 h. It was speculated that OD600 reached the maximum after 25.5 h, which indicated that the growth of pTRIP-ccdB was not affected by the toxic protein ccdB at 37 °C. At the same time, it may indicate that the concentration of AI is less than 1mmol, and pTRIP-ccdB is not affected by the toxic protein ccdB at 37 degrees.

1. We used pTRIP-EGFP to verify the feasibility of the temperature control switch and the effect of different temperatures on the temperature control system ( pTRIP-EGFP ). The results showed that the fluorescence intensity of EGFP was the largest at 22.66 degrees ; at the same time, it can be seen that in 16-22.66, EGFP showed an upward trend, indicating that the 16-22.66 degree temperature control switch gradually opened with the temperature ; when the temperature exceeds 22.66-37 degrees, the temperature control system is gradually closed. It is of great value to our project. It can not only prove the feasibility of the temperature control system, but also provide data support for the sensitivity adjustment of the temperature control switch. In the future, the temperature range can be set to 22-26 degrees, we can further explore the impact of temperature on the temperature control system.

2. We tested the function of the temperature-controlled switch pTRIP-ccdB in E.coli DH5α, and we wanted to find the optimal induction concentration and time for the growth ability ( OD600 ) of pTRIP-ccdB by modeling.At 22 degrees, the minimum OD600 was 0.0981, the corresponding AI concentration was 0.87, and the time was 2.5 h . At 37 degrees, the maximum OD600 was 1.3762, the corresponding concentration was 1.0mmol, and the time was 25.5h ; this shows that at 22 degrees, toxic protein expression inhibits colony growth, while at 37 degrees, the strain can grow normally, providing data support for adjusting the sensitivity of the temperature control system. In the future, pTRIP-ccdB can be set at different temperatures to explore the effect of different temperatures on the growth ability of pTRIP-ccdB.