Team:Shanghai HS ID/Engineering
Engineering
Background
Lactobacillus casei is commonly used in the production of cheese, yogurt, and other
products. Studies have shown that L. casei can regulate the intestinal flora, lower blood pressure and
cholesterol, enhance immunity, prevent cardiovascular disease and fight cancer. The unique probiotic
function of L. casei and its wide application in the field of food and medicine make the study of the
molecular mechanism of L. casei have great significance, and the in-depth study of it involves the transfer
of foreign DNA into L. casei.
Research showed that the transfermation efficiency of foreign DNA in L. casei is
much lower than the model microorganism Escherichia coli. This will restrict the research on the molecular
mechanism of L. casei. Therefore, in order to realize the heterologous expression of foreign genes, it is
first necessary to realize the efficient transformation of L. casei.
Design
In this study, the CRISPR-Cas9 gene editing method was used to knock out the
restriction enzyme LSEI-2094 gene of L. casei ATCC 334. The gene knockout strains were selected by
antibiotics resistance, PCR and sequencing. The yield L. casei strain is of great significance to the
development of L. casei genetic engineering.
Figure 1 shows the design of a CRISPR-Cas9-based gene knockout vector in L. casei
ATCC 334. Single plasmid CRISPR-Cas system is applied, namely gRNA, Cas effector protein, and repair
template on one plasmid.
Figure 1. CRISPR-Cas9-based gene knockout vector design in Lactobacillus casei ATCC
334. P is the promoter (promotor); T is the terminator (terminator); pNCas9 is the Cas effector protein;
HA-L and HA-R are the left homology arm and the right homology arm, respectively.
Build
Figure 2. A: DNA profile of the plasmid PLCNICK; B: DNA Profile of LSEI-2094+
upstream and downstream homologous arms.
Test
(1) Improvement of transformation efficiency
Plasmid pIB165 was transformed the as the foreign DNA to test the transformation
efficiency of our modified L. casei. It took several days to culture and finally observed the comparison
results. As shown in Figure 3, the modified L. casei (KO) has higher transformation efficiency with
remarkably more colonies than the wild strain.
Figure 3. Comparison between the wild L. caseil (left) and modified L. casei
(right, LSEI-2094 knocked out) in transformation.
Figure 4. Histogram comparison between wild strain and KO strain.
In addition, we measured OD600 of these strain groups which were pre-spreaded
plates with different volumes of bacteria solutions so as to quantify the transformation results as showing
above (Figure 4).
(2) model build
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.
According to the scatter plots, we chose to use the quadratic polynomial equation
to
build the model :
After calculation, below are the constants of the solved quadratic polynomial
equations
of the KO group and Wild group, respectively.
Table 2. Model results
Figure 5. Comparison between two fitting curves of KO group(red) and Wild group(blue).
In figure 5, 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.
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:
Learn
In conclusion, it indicates that our modified L. casei has much higher efficiency
of
the foreign plasmid transformation than the wild and the modified L. casei has great potential to be used as
the
recombinant carrier in various areas.