Purpose

Given that GST-VP1-LTB proteins showed weak expression, we needed to optimize the expression conditions in terms of the concentration of IPTG solution and the growth period of E.coli. The amount of protein expression will be integrated with the above two factors, in order to predict the optimal protein expression condition of EV71-VP1 and EV71-VP1-LTB, by the means of modeling.

Initial data

After transforming pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB into E.coli BL21, We harvested the bacteria solution of different concentration at different time(OD₆₀₀ =0.5/0.6/0.8/1.0), separated each group into two, and induced each of the two groups with 1mM IPTG and 10mM IPTG respectively. Then we ran the PAGE gel of all the groups and stained the gel with coomassie brilliant blue staining solution. The strongest band in each group corresponded to GST, GST-VP1 and GST-VP1-LTB respectively.

Figure 1: PAGE gel after Coomassie Brilliant Blue staining

To compare the expression of proteins in each group, we used the software ImageJ to quantify the brightness of the selected bands as gray value. We collected the gray values and arranged them into a table.

Model Construction

Integrating the results obtained in the table above, we used the software Mat lab to construct a model.

In order to analyze the experssion factors, we divided the data into four groups in terms of the types of proteins and the concentration of IPTG solution: VP1(1mM IPTG), VP1(10mM IPTG), VP1-LTB(1mM IPTG), VP1-LTB(10mM IPTG). And we took OD₆₀₀ of the bacteria solution as the x-axis and the gray value as y-axis.

IPTG = 1mM

After trying different types of data equations, such as linear equation, polynomial equation and exponential equation, we chose the polynomial equation, y=p₁x³+p₂x²+p₃x+p₄ which fits our experiment results the most to construct the model about the relationship between the OD₆₀₀ of our bacteria and the Gray value (the protein expression level).

1. VP1 Group

Figure 2. The fitting parameter results of y=p₁x³+p₂x²+p₃x+p₄ model when IPTG=1mM for VP1 group

Figure 3. The fitting curve of y=p₁x³+p₂x²+p₃x+p₄ model when IPTG=1mM for VP1 group

As showing above in Figure 2, the fitting degree of the polynomial model reaches 1, which you could also tell from the smooth curve in Figure 3. Based on this model, we could predict the gray value, namely the protein expression level of the engineered strain (VP1) once the OD₆₀₀ value (the growth period) is given.

2. VP1-LTB Group

Figure 4. The fitting parameter results of y=p₁x³+p₂x²+p₃x+p₄ model when IPTG=1mM for VP1-LTB group

Figure 5. The fitting curve of y=p₁x³+p₂x²+p₃x+p₄ model when IPTG=1mM for VP1-LTB group

Figure 6. Comparison diagram of the fitting curves of VP1 group and VP1-LTB group when IPTG=1mM

For VP1-LTB group when IPTG=1mM, we can predict that the gray value (the protein expression level) could reach the highest when OD₆₀₀ is 0.9. According to figure 6, it indicates that LTB could promote the ptotein expression level of the engineered bacteria in the higher OD₆₀₀ range (> 0.69), namly the late growth period.

IPTG = 10mM

We still use the polynomial equation to fit the relationship between the OD₆₀₀ value of the engineered strains ( VP1, VP1-LTB ) and the gray value when IPTG=10mM. The fitting parameter results and the fitting curves are showing below.

1. VP1 Group

Figure 7. The fitting parameter results of y=p₁x³+p₂x²+p₃x+p₄ model when IPTG=10 mM for VP1 group

Figure 8. The fitting curve of y=p₁x³+p₂x²+p₃x+p₄ model when IPTG=1mM for VP1 group

According the figure 7 and 8, the polynomial fucntion fits these data quite well with the fitting degress 1. Compared to the decline trend in figure 3, there are a peak (OD₆₀₀=0.9) and a trough (OD₆₀₀=0.64) when the IPTG concentration goes up to 10 mM.

2. VP1-LTB Group

Figure 9. The fitting parameter results of y=p₁x³+p₂x²+p₃x+p₄ model when IPTG=10mM for VP1-LTB group

Figure 10. The fitting curve of y=p₁x³+p₂x²+p₃x+p₄ model when IPTG=10mM for VP1-LTB group

Figure 11. Comparison diagram of the fitting curves of VP1 group and VP1-LTB group when IPTG=10mM

For VP1-LTB group when IPTG=10mM, the overal trend of the grey value (the protein expression level) is monotonically inceasing by the OD₆₀₀ (figure 10). When the concentration od IPTG is 10 mM, we could see that basically the gray value of VP1 is higher than that of VP1-LTB group until the OD₆₀₀reaches 0.97. So herein we can draw a similar conclusion that LTB could promote the ptotein expression level of the engineered bacteria in the very late growth period (OD₆₀₀ >0.97).

According to all the above, we will suggest the optimum stage of our engineered bacteria for the protein expression will be the OD₆₀₀ 0.8~0.9. For the concentration of IPTG, it seems not that relevant. For the VP1 group, 10mM could induce the protein more. But for the VP1-LTB group, 1mM could promote much stronger in the late period.

Future Plan

However, due to the difficulty of the protein expression in L.casei, we would need to optimize the incubation and electroporation of L.casei or reconstruct the plasmids. In addition, our partner Pro-casei team will continue their development of modifying L. casei.

If we succeed in stage 2 where the engineered L. casei could express the VP1 protein optimum, it would be more promising that we could reach the next big stage - let the mice take the transformed L.casei via gavage, and then measured the level of anti-VP1-LTB IgG in the serum of the mice and the level of IgA in their feces.