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The smaller the MIC value, the lower concentration will be required to inhibit S.mutans growth. It shows that the inhibiting effect of antimicrobial peptide is better. According to Table.1, the MIC of TP-1 is 8μg/mL means that these two antibacterial peptides have a high inhibit effect on S.mutans. TP-1 was selected to develop into prevention and treatment products for dental caries.

Construction of the antimicrobial Peptides Multimer (AMPM)

Since TP-1 is composed of only a few amino acids, it is difficult to biosynthesize directly. Firstly, antimicrobial Peptides Multimer was constructed. TP-1M containing 73 amino acids was joined by E, cleavage sites of Glu-C. The amino acid sequence of TP-1M is shown in Fig.1.

Fig.1 Amino Acid Sequences of TP-1M

In order to further improve the inhibitory effect of TP-1 on S.mutans, we designed a specific targeting TP-1 sequence (BTP-1) based on the signal molecule CSP in the quorum sensing signaling system of S.mutans. In order to achieve the expression of BTP-1, we used E as the connection site to form a specific targeting antimicrobial peptide polymer (BTP-1M). The amino acid sequence of BTP-1M is shown in Fig.2 below.

Fig.2 Amino Acid Sequences of BTP-1M

Construction and Identification of Recombinant Expression Vector of antimicrobial peptides

We cloned the TP-1M gene into the pGEX-4T-2 expression vector. Positive clones were selected from LB plates containing 100μg/mL ampicillin for restriction analysis. The results show that the fragment size is the same as TP-1M, shown in Fig.3. Hence, the recombinant expression vector pGEX-4T-2-TP-1M was successfully constructed.

Snapgene® diagram of pGEX-4T-2-TP-1M

Fig.3 1% agarose gel image showing the results of PCR and double digestion

M,D2000 Plus Marker;Line 1~2, pGEX-4T-2-TP-1M double restriction digestion verification

Snapgene® diagram of pGEX-4T-2- BTP-1M

Fig.4 1% agarose gel image showing the results of PCR and double digestion

M,D2000 Plus Marker;Line 1~3, pGEX-4T-2-BTP-1M double restriction digestion verification;

Expression of GST-TP-1M/GST-BTP-1M in E.coli BL21

The recombinant strain E.coli BL21 (DE3) with pGEX-4T-2-TP-1M vector was induced by IPTG for 6 hours and then collected for SDS-PAGE and Western blot analysis. The results of SDS-PAGE were shown in Fig.5. After IPTG induction, the E.coli BL21 transferred with pGEX-4T-2 vector produced 26kDa GST protein (lane2) and E.coli BL21 transferred with pGEX-4T-2-TP1M produced 36kDa protein matched well with our GST-TP1M fusion protein (lane 4). In contrast, Non-induced E.coli BL21 cells did not express GST or GST-TP-1M (lane 1, lane 3). Western blot results showed 36kDa fusion protein carry GST tag (line 5). The results showed that the constructed tandem TP-1M could be expressed in the fusion expression system.

Fig.5 SDS-PAGE analysis of GST-TP-1M expression in E.coli BL21

Lane 1, E.coli BL21 (pGEX-4T-2) without IPTG inducing; Lane 2, E.coli BL21 (pGEX-4T-2) with IPTG inducing; Lane 3, E.coli BL21 (pGEX-4T-2-TP-1M) without IPTG inducing; Lane 4, E.coli BL21 (pGEX-4T-2-TP-1M) with IPTG inducing; M, Marker; Lane 5, western blot

The recombinant strain E.coli BL21 (DE3) with pGEX-4T-2-BTP-1M vector was induced by IPTG for 6 hours and then collected for SDS-PAGE and Western blot analysis. The results of SDS-PAGE were shown in Fig.6. After IPTG induction, the E.coli BL21 transferred with pGEX-4T-2 vector produced 26kDa GST protein (lane2) and E.coli BL21 transferred with pGEX-4T-2-BTP-1M produced 44kDa protein matched well with our GST-BTP-1M fusion protein (lane 4). In contrast, Non-induced E.coli BL21 cells did not express GST or GST-TP1M (lane 1, lane 3). Western blot results showed 44kDa fusion protein carry GST tag (line 5). The results showed that the constructed tandem BTP-1M could be expressed in the fusion expression system.

Fig.6 SDS-PAGE analysis of GST-BTP-1M expression in E.coli BL21

M, Marker;Lane 1, E.coli BL21 (pGEX-4T-2) without IPTG inducing; Lane 2, E.coli BL21 (pGEX-4T-2) with IPTG inducing; Lane 3, E.coli BL21 (pGEX-4T-2-BTP-1M) without IPTG inducing; Lane 4, E.coli BL21 (pGEX-4T-2-BTP-1M) with IPTG inducing; Lane 5, western blot

Fusion protein purification results

Recombinant bacteria were induced to express, and the cells were collected, ultrasonically broken, and the inclusion bodies were dissolved. After affinity chromatography, the fusion proteins GST-TP1M was obtained. The SDS-PAGE electrophoresis detection result is shown in the Fig.7.

Fig.7 SDS-PAGE analysis of expression and purification of GST-TP1M in E.coli BL21

M, Marker;Lane 1, E.coli BL21 (pGEX-4T-2) without IPTG inducing; Lane 2, E.coli BL21 (pGEX-4T-2) with IPTG inducing; Lane 3, E.coli BL21 (pGEX-4T-2-TP-1M) without IPTG inducing; Lane 4, E.coli BL21 (pGEX-4T-2-TP-1M) with IPTG inducing; Lane 5, Broken supernatant stock solution; Lane 6, Effluent; Lane 7, Rinsing liquid; Lane 8, Eluent

Recombinant bacteria were induced to express, and the cells were collected, ultrasonically broken, and the inclusion bodies were dissolved. After affinity chromatography, the fusion proteins GST-BTP-1M was obtained. The SDS-PAGE electrophoresis detection result is shown in the Fig.8.

Fig.8 SDS-PAGE analysis of expression and purification of GST-BTP-1M in E.coli BL21

M, Marker;Lane 1, E.coli BL21 (pGEX-4T-2) without IPTG inducing; Lane 2, E.coli BL21 (pGEX-4T-2) with IPTG inducing; Lane 3, E.coli BL21 (pGEX-4T-2-BTP-1M) without IPTG inducing; Lane 4, E.coli BL21 (pGEX-4T-2-BTP-1M) with IPTG inducing; Lane 5, Broken supernatant stock solution; Lane 6, Effluent; Lane 7, Rinsing liquid; Lane 8, Eluent

Expression of prepared antimicrobial peptide activity results

After the purified fusion protein is digested by thrombin and glutamate endopeptidase, antimicrobial peptide monomers are obtained. The antibacterial results are shown in the Fig.9, the purified TP-1 has a good inhibitory effect on S.mutans.

Left:ddH2ORight:TP-1M

Fig.9 Isolation and purification of antimicrobial peptide inhibition zone

Expression of TP-1 in lactic acid bacteria

Since E.coli fusion expression requires complex protein purification to obtain the desired peptide, there are uncontrollable factors in the separation process (such as sample contamination). It is difficult for the product to reach food grade, so we plan to use lactic acid bacteria as the chassis to directly secrete and express the antimicrobial peptide hydrolysis to play a role, so that the secretion expression system reduces the complicated separation and purification process.

Construction of Recombinant Plasmid pMG36N- TP-1M

The oral cavity is in an acidic environment when the human body suffers from mild dental caries. Therefore, the acid-inducible promoter P170 was used for TP-1M expression and secretion signal peptide SPusp45 were used to secrete the target protein directly. The construction of lactic acid bacteria expression system was referred to 2019 SZPT-CHINA project. The recombinant vector named pMG36N-TP-1M and was confirmed by PCR analysis. As shown in Fig.10, the fragment size is the same as TP1M. The results showed that the recombinant expression vector pMG36N-TP1M was successfully constructed.

Fig.10 1% agarose gel image showing the results of PCR and double digestion

M,D2000 Plus Marker;Line 1~12, Colony PCR screening of recombinants;Line 13, Negative control.

Chewing gum to prevent dental caries

We added the expressed antimicrobial peptides to chewing gum for inhibiting the growth of S.mutans. We have also established a method for preparing probiotic chewing gum that lays the foundation for the subsequent preparation of probiotics that continue to express antimicrobial peptides into chewing gum products to prevent dental caries.(See the detail,click here)

Part Ⅱ Detection system for Streptococcus mutans

Construction and Identification of detection system based on comD and comE induction of S.mutans

The pelB-comD, comE and nlmC-sfGFP genes were cloned into pET28a(+) vector. Positive clones were selected from LB plates containing 50μg/mL kanamycin sulfate for double restriction analysis and PCR analysis. As shown in Fig.11-13, the fragment size is the same as pelB-comD, comE and nlmC-sfGFP. The results showed that the recombinant expression vector pET28a(+)-pelB-comD-comE-nlmC-sfGFP (pET28a-DEG) was successfully constructed.

Fig.11 1% agarose gel image showing the results of PCR and double digestion

M,1 Kb Marker; Line 1~3, comD PCR verification; Line 4~6, comD double restriction digestion verification.

Fig.12 1% agarose gel image showing the results of PCR and double digestion

M,1 Kb Marker;Line 1~3, nlmC-sfGFP PCR verification;Line 4~6, nlmC-sfGFP double restriction digestion verification

Fig.13 1% agarose gel image showing the results of PCR and double digestion

M,1 Kb Marker;Line 1~3, comE PCR verification;Line 4~6, comE double restriction digestion verification

Snapgene® diagram of pET28a(+)-DEG

The result of the detection system on CSP induction

The recombinant strain E.coli BL21 (DE3) with pET28a-DEG vector works as a detector. Different concentrations of CSP were added to the culture medium for checking the output variations. When the concentration of CSP reaches 0.5mg/ml, the comD-CSP complex would Phosphorylation of comE, phosphorylated comE and nlmC promoter combined to start the expression of the downstream fluorescent gene sfGFP.（Fig.14). The fluorescence intensity gradually increased as the culture time prolonged. After 24h, there was a significant difference in fluorescence intensity. This result showed that the density sensing system in E.coli detector was successfully functional.

Fig.14 The relationship between CSP concentration, time, and fluorescence intensity

The detection result of S.mutans using E.coli detector

To verify whether the constructed E.coli detector can directly detect S.mutans. The cultured S.mutans was collected and then added to E.coli detector culture for co-cultivation. After co-culturing for 24h, there was no significant difference in fluorescence intensity, and results were unsuccessful. The result showed that co-cultivation should be optimized because the medium and aerobic condition was different for these two bacteria. Therefore, the detection of S.mutans may require the development of another detector.

Fig.15 Co-cultivation of engineered bacteria and S.mutans.

Construction of a visible light E.coli detector using β-galactosidase as the signal generator

The sfGFP（Super folded green fluorescent protein）was used as the signal generator for the E.coli detector, and the fluorescence can not be observed until to illuminate sfGFP by excitation light. The sfGFP gene was replaced with lacZ gene to make it simple that was encoded by β-galactosidase. When the concentration of CSP reaches 0.5mg/ml, the E.coli detector will express β-galactosidase. Then the subtract X-gal in the medium will be catalyzed to blue product; the medium will show a visible color change.

Fig.16 Detection of engineering bacteria to hydrolyze X-gal

Implementation of POCT system using E.coli detector

Based on the marketing research results, we designed a POCT test kit using our E.coli detector. In this kit ,we keep the broth in the detection tube, and put the dry powder of E.coli detector into the lid of the detection tube. We have a special design for the lid. When using, press the button on the lid to press the dry powder into the broth in the tube .The testing result showed that we Successfully cultivated the flora using our POCT test kit and device.

Analysis of Suicide mechanism

Recently, we have constructed a suicide mechanism to ensure that the transgenic bacteria will not be harmful to the environment. A suicide mechanism is regulated by the lysine riboswitch based on the difference between the human oral environment and the lysine environment in the external environment. When the bacteria are colonized in the oral cavity, the riboswitch controlled by lysine is turned off. When the bacteria escape the oral environment, the riboswitch is turned on to exhibit the toxic protein RalR, a non-restrictive endonuclease that can cut DNA by killing bacteria that escaped the human body.

The results of the construction of the recombinant plasmid pET28a-RS-RalR are shown in Fig.18. The results of E.coli BL21 with pET28a-RS-RalR after induction of recombinant bacteria by different lysine concentrations are shown in Fig.17. The results showed that when the concentration of glucose in the environment was less than 25um, the growth of recombinant bacteria was inhibited significantly.

Fig.17 1% agarose gel image showing the results of PCR and double digestion

M,D2000 Plus Marker; Line 1~3, pET28a（+）-RS-RalR double restriction digestion verification; Line 4~6，RS-RalR PCR verification

Lysine riboswitch

We put the lysine riboswitch connected sfGFP engineered bacteria in different concentrations of lysine for 24 hours, and then tested the fluorescent expression of the bacteria. The results are shown in Fig.18. The bacteria are in the lysine at a concentration of 30μmol/mL. Fluorescence expression was significantly inhibited, indicating that the lysine riboswitch can work normally.

Fig.18 Relationship between Lysine Concentration and Fluorescence Intensity in Lysine Riboswitch

Toxin inhibitory result

We streaked the engineered strain into a 1Mm IPTG plate for overnight culture, and observed that the engineered strain containing the RalR gene did not grow on the plate. The result is shown in Fig.19;

Fig.19 Safety switch function verification tablet result

Team:SZPT-CHINA/Results