Team:Shanghai HS ID/Model

Shanghai_HS_ID

MODEL

In order to scientifically determine the transformation efficiency of our modified L. casei (KO) and the wild L. casei (Wild), we collected the colony cultured which were pre-spread plates with different volumes of bacteria solutions and measured their OD₆₀₀ after cultured for the same hours. In the meantime, we also aimed to explore the optimal condition for our modified L. casei’ growth by applying different amounts of the bacteria seed solution.

Table 1. OD₆₀₀ of cultured L. casei

According to the scatter plots, we chose to use the quadratic polynomial equation to build the model :

The coding we used is given below:

“
clear;clc;
v0=[50 100 150];
od10=[0.035 0.289 3.84];
od20=[0.255 1.984 5.21];
p1=polyfit(v0,od10,2)
p2=polyfit(v0,od20,2)
v=[50:150];
od1=polyval(p1,v);
od2=polyval(p2,v);
plot(v,od1,'b','LineWidth',2)
hold on
plot(v,od2,'r','LineWidth',2)
plot(v0,od10,'k*','LineWidth',2)
plot(v0,od20,'g*','LineWidth',2)
hold off
”

After calculation, below are the constants of the solved quadratic polynomial equations of the KO group and Wild group, respectively.

Table 2. Model results

Sample	p1	p2	p3
KO	0.0007	-0.0938	3.0780
Wild	0.0003	-0.0103	0.0230

Figure 1. Comparison between two fitting curves of KO group(red) and Wild group(blue)

Conclusion

In figure 1, we can clearly see that the modified L. casei shows much higher transformation efficiency than the wild L. casei especially when the volume of the initial bacteria seed solution is used less than 100 uL when the difference between them is remarkable.

Solution

We constructed a plasmid equipped with a CRISPR-Cas9 complex that cuts and removes a selected segment of the L. casei DNA. Our target gene is LSEI-2094. This gene is involved in the synthesis of an enzyme that is essential in the restriction-modification system [5]. The cell's efforts to repair its DNA induces mutations, which help to inactivate the gene and prevent the production of the enzyme. After this modification foreign DNAs could enter and express themselves much more efficiently in L.Casei.With a strain of bacteria ready to be tailored to our needs, the benefits are immeasurable. The benefits of L. caseicould come to fruition if the technology is applied to popular and impactful industries such as pharmaceuticals and food.

Besides, the equation model we built for the modified L. casei as showing below, could be used to analyze the relationship between the volume of the bacteria seed solution and its OD₆₀₀, it could be used as a reference when we conduct the expression efficiency tests in the future. If we could further build the relationship between the expression level and OD₆₀₀, we could adjust the volume of the bacteria seed solution for culturing accordingly.

Model for the modified L. casei: f(x) = 0.0003x³ - 0.0103x² + 0.0230