Team:Shanghai HS ID/Partnership
Partnership
Inspiration and Common Goal
Lactobacillus casei is commensal bacteria that have long
been utilized as probiotics and are generally regarded as safe for use in humans, making them prime
candidates for vector design. Recent research has, however, focused on the probiotic properties rather than
their specific interactions with the mucosal immune system.
In order to rationally develop L. casei as vaccine vectors,
EV71 terminator and us decided to partner with each other in developing the HFMD Oral Vaccines using L.
casei as a substitute for E. coli. EV71 terminator will construct plasmids containing DNA expressing the
protein coat of the virus, and convert the plasmids into the experimental strain of L. casei cultivated by
us. The wild type of L. casei exhibits low expression rates of plasmids, since it is protected by the
Restriction Modification system, which destroys foreign DNA and prevents its expression in the L. casei
bacteria. We design to remove a specific segment of L. casei’s DNA which codes for a pivotal enzyme
participating in the Restriction Modification system. Hopefully, this will create a strain of new L. casei
with higher expression rates along with its immense health benefits.
Experiment Design
Figure 1. Visual Representation of Experimental Design from EV71 Terminator
EV71 terminator designed to introduce the constructed
plasmid into E.coli to verify the viability of the concept of producing oral vaccines. The plasmids were
then transformed into L.casei to detect for the correct protein expression.
For plasmid constructions, they first conducted PCR to
obtain the VP1, VP1-linker and LTB fragments, then OE PCR to obtain VP1-LTB fragments. The VP1 fragments,
VP1-LTB fragments and pGEX-6P-1 will undergo restriction enzyme double digestion and ligation to finally
construct the expected plasmids, pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB, which would be shortly transformed
into E. coli BL21 as well as L. casei ATCC334 for the antibody expression. To evaluate the performance of
the protein expression, they conducted the SDS-PAGE procedure and the Western Blot experiments, combined
with Coomassie Brilliant Blue Staining as function tests.
Figure 2: Visual Representation of Experimental Design from Pro-CASei
For us Pro-CASei, we plan to utilize CRISPR-Cas9 to reform
wild Lactobacillus. casei by knocking out the gene LSEI-2094 in order to improve the conversion and
expression efficiency of exogenous genes.
We obtained sgRNA with pLCNICK as the template by PCR. In
the same way, the upstream and downstream homologous arms could be obtained with L. casei as the template.
After the ligation experiment, sgRNA, the upstream and downstream homologous arms were inserted into pLCNICK
to be the expected plasmid. Initially, we transformed the plasmid into E. coli DH5α for culturing and then
it will be sent for sequencing after the plasmid extraction. Once we obtain the correct plasmid from EV71
terminator, we would introduce it into the L. casei ATCC334 by electrotransformation in order to knock out
gene LSEI-2094.
In the final stage where we obtained the LSEI-2094 gene
defect L. casei ATCC334, we evaluated its performance by transferring and expressing a foreign plasmid -
pIB165 with the wild L. casei ATCC334 as a comparison.
By cooperating, the two teams will design an experiment to
transform the plasmid constructed by EV71 Terminator before implanting it into the transformed colony of L.
casei bacteria which was modified by us.
Progress and Collaboration
EV71 Terminator
Figure 3. PAGE gel of GST, GST-VP1 and GST-VP1-LTB under different expression conditions in E.
coli
Figure 4. PAGE gel of GST, GST-VP1 and GST-VP1-LTB after staining in L. casei ATCC334
Firstly they successfully obtained the expected plasmids,
pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB, which were proved by colony PCR and sequencing. Besides, these plasmids
were successfully transformed into E.coli BL21 and L.casei ATCC334 which were also verified by colony PCR,
respectively.
After that, they transformed them into E.coli BL21 and L.
casei ATCC334 to verify the protein expression by SDS PAGE with Coomassie Brilliant Blue Staining and
Western Blot, respectively, to stain the bands. By selecting bacteria solutions of different concentrations
and inducting them with IPTG solutions of different concentrations, EV71 terminator determined the optimum
conditions under which the proteins GST-VP1 and GST-VP1-LTB were expressed better. In addition, it indicated
that LTB could promote the expression of VP1 during the late growth period of E.coli.
However, there was no obvious band in certain KD ranges of
the L. casei PAGE gel (figure 4), which meant there was no obvious expression of GST-VP1 and GST-VP1-LTB in
L. casei, which is probably because of the restriction-modification system in wild L. casei. Therefore, it
is necessary to modify the L. casei and the task of our team is significant.
Pro-CASei
Figure 5. results from Pro-CASei
Toughing through several failures, we successfully
constructed the plasmid pNCas9-LSEI-2094, the core task which could be used for the later Gene Knock-out
experiment. During the time, we also prepared L. casei competent cells and MRS media which were also
provided to EV71 Terminator to continue their experiments.
After electrotransforming into L.casei ATCC334, it took
several days for culture and those strains were verified as positive by sequencing. In the next stage, we
decided to test the performance of our modified L. casei by being transformed with a foreign plamid pIB165
meantime comparing to the wild L. casei. As showing above, we can see that the modified L. casei (KO) has
higher transformation efficiency than the wild (Wild).
Collaboration
EV71 Terminator
They explored and practiced the transformation incubation procedures of L.casei, an
elementary step of the
functional test designed by Pro-CASei. These protocols helped us perfect the experiment procedures, and laid
down the foundation for further steps. Besides, their attempt to use the wild L. casei could serve as a
comparison to our modified L. casei in the future.
Pro-CASei
We supplied EV71 Terminator with necessary materials, such as the L. casei
competent cells and MRS media to cultivate both the wild strain and experimental strain of L. casei. This
simplified the complex experiment procedures and accelerated the project process.
Other Collaboration
We two teams published articles on our official accounts introducing each other’s
team, topics, and future plans.
Challenge and Future Plan
Even though the EV71 Terminator’s carrier pGEX-6P-1 is
generally said to be able to express in Lactic Acid Bacteria, no evidence specifies its viability in
L.casei. Possible reasons behind the scarce research on L. casei can be the relatively low electroporation
efficiency, slow growth rate, and the Restriction Modification system in L.casei. These are all challenges
that the two teams together will need to tackle in the future to transform the plasmid containing VP1-LTB
into the new experimental strain L. casei which would be pre-modified and cultivated by us.
Currently, EV71 terminator has verified the feasibility of
the oral vaccine and determined the optimum condition for the protein expression in E.coli. Further steps
would be to substitute E. coli with L.casei to improve the vaccine and protect patients from the nasty
effects of bacteria imbalance.
Now we had obtained the modified L.casei and would conduct
more functional tests to evaluate the performance.
Because of the delicate conditions required for optimized
protein expression, the two teams will either need to improve the incubation and electroporation techniques
or reconstruct the plasmids to maximize the expression of GST-VP1-LTB in L. casei.
No doubt that both teams strive to improve the
transformations rate of the foreign plasmids to L. casei than that seen in the wild-type bacteria, allowing
us two teams to step closer to our common goal - HFMD Oral L. casei-Based Vaccines.
Assuming we two teams are able to detect a higher
expression rate in the L. casei, the next step would be to gavage feed the transformed L. casei to groups of
mice and measure the level of anti-VP1-LTB IgG in their serum and the level of IgA in their feces. With
statically significant differences between the levels of the experimental group mice and control group, this
experiment might have profound evidence to prove whether immunogenicity in the mice that consumed the
transformed L. casei. In other words, this will prove the feasibility of the whole project.
Reference:
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intraepithelial neoplasia grade 3 (CIN3) elicits E7-specific mucosal immunity in the cervix of CIN3
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[3] Adachi K , Kawana K , Yokoyama T , et al. Oral immunization with a
Lactobacillus casei vaccine expressing human papillomavirus (HPV) type 16 E7 is an effective strategy to
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