Team:Shanghai United HS/Engineering
Engineering
Introduction
Dental caries is a common disease. It not only directly affects human oral health,
but also often causes adverse symptoms in other parts of the body. Global disease statistics in 2016 show
that the incidence of dental caries in the population is ranking second among common diseases. Studies have
shown that the formation of dental plaque is the result of the joint action of a variety of bacteria,
including Streptococcus mutans, Lactobacillus, Actinomycetes, etc. Phage lyase ClyR (combined from different
bacteriophage lytic enzymes) has a broad bactericidal spectrum, especially the only one reported to be
extremely strong against Streptococcus mutans and Streptococcus mulberry. The enzyme is promising to kill
these two kinds of streptococci are the main cause of dental caries.
Design
This project designed a genetically engineered probiotic (Escherichia coli strain
Nissle 1917 (EcN)), which can be induced and expressed of lysate enzyme ClyR upon exposed to monosaccharides
(arabinose) which released from food arabinoxylan in the oral (Figure 1).
Figure 1 Schematic diagram of ClyR in the prevention or treatment of dental caries.
Build
Genetically engineered probiotics bacteria which produce and secret ClyR are
constructed. Construct puc57-kan-mini-J23101-OmpA-arab-TT secretes arabinosidase to hydrolyze
polysaccharides into monosaccharides; construct B pBAD-Myc-HisA-OmpA-ClyR-6His-TT is induced by arabinose
and produce the ClyR enzyme. Construct C pBAD-Myc-HisA-OmpA-amilGFP-TT is used to test the function of the
system. All the constructs were confirmed by DNA sequencing.
Figure 2. Schemtic maps of plasmids A, B, and C..
Test
(1) AmilGFP expression in cells transformed with plasmid C
In the cells transformed with plasmid C, the fluoroscopic data of cells increased
with the addition of arabinose. Meanwhile, the fluoroscopic data become larger with the prolongation of
incubation time.
Figure 3. The fluoroscopic data of cells transformed with plasmid C with the addition of
arabinose.
(2) AmilGFP expression in cells transformed with plasmids A and C
In the cells transformed with constructs A and C, the fluoroscopic data of cells
increased with the addition of araboxylan. Meanwhile, the fluoroscopic data become larger with the
prolongation of incubation time (Figure 4).
Figure 4. The fluoroscopic data of cells transformed with plasmids A and C.
(3) ClyR expression
Plasmid A and plasmid B were co-transformed to E. coli Nissle 1917 by
electroporation. The araboxylan with final concentration 0, 0.3%, 0.6%, 1.0%, 1.5% and 2.0% (w/v) was added
into the culture to induce ClyR expression. For the comparison, E. coli Nissle 1917 with plasmid B was
cultured by the same way and the expression of ClyR was induced by the addition of arabinose with final
concentration of 0 μM, 10 μM,30 μM,0.1 mM, 0.2 mM,0.5 mM and 2 mM.
The protein concentration was monitored at 595 nm using Multiscan Spectrum
(BioTek). Read the data for three times, recorded the average of the A595 data (Table 1). The A595 of the
culture supernatant of E. coli with plasmid A and B suggested the possible expression of ClyR (Table 2). The
A595 data at “0 %” was referred as blank, and the protein concentration could be calculated referred to the
standard formula.
Table 2. The protein concentration of the culture supernatant of E. coli with
plasmids A and B.
As can be seen from figure 5, in the transformed bacteria with plasmid A and B, the
concentration of the reducing sugar increased with the elongation of the incubation time. During the
expression process, bacteria lysis occurred with the elongation of the incubation time. But the ClyR band
could obviously be obtained as is shown in figure 5.
Figure 5. The SDS-PAGE of supernatant.
(4) In vitro activity assay of ClyR
E. coli Nissle 1917 transferred with plasmid A and B were culture with
Gram-positive bacteria Lactobacillus casei subsp. casei (ATCC334). The value of OD600 was monitored using
Multiscan Spectrum (BioTek). The OD600 of the L. casei subsp. casei cell suspension gradually reduced with
the addition of supernatant (Figure 6).
Figure 6. The in vitro assay of ClyR.
Learn
AmilGFP was successfully expressed, suggesting the feasibility and possibility of
the inducible secretory expression of ClyR. We have successfully obtained the ClyR protein in the culture
supernatant of cells carrying plasmids with araB and ClyR genes. The concentration of ClyR was low, so more
efforts are still needed in future. It is worth to note that the expression of ClyR protein may cause the
host to suicide, which can be supported by the obvious cell lysis and the presence of flocculent
precipitate.