Part:BBa_K4030009

Myc-HisA-OmpA-ClyR-6His-TT

Usage and Biology

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.

Figure 1 Schematic diagram of ClyR in the prevention or treatment of dental caries..

Construct design

The ClyR is under araBAD promoter, which is induced by arabinose. And ClyR is linked with ompA and this sequence is inserted into plasmid (Figure 2 and 3).

Figure 2. Phage lyase ClyR expression system..

Figure 3. Schematic map of Phage lyase ClyR plasmids..

The profiles of every basic part are as follows:

BBa_K4030005

Usage and Biology

BBa_K4030005 is a coding sequence of from Streptococcal phage. Phage lyase ClyR is promising to kill he main cause of dental caries .

BBa_K4030010

Usage and Biology

The araBAD promoter of the L-Arabinose operon of Escherichia coli allows tightly controlled, titratable expression of your protein through the regulation of specific carbon sources such as glucose, glycerol, and arabinose. pBAD is ideal for expressing toxic proteins and optimizing protein solubility in E. coli.

BBa_K4030000

Usage and Biology

Outer membrane protein A (OmpA) is a major protein in the Escherichia coli outer membrane.

BBa_K4030003

Usage and Biology

It is an epitope tag derived from c-myc gene

BBa_K4030006

Usage and Biology

It is a polyhistidine tag, which is used in the purification of recombinant proteins

Experimental approach

Plasmid A (puc57-kan-mini-J23101-OmpA-araB-TT) and plasmid B (pBAD-Myc-HisA-OmpA-ClyR-6His-TT) 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.

Gels were scanned with the ImageQuant™ LAS 4000 mini (GE Healthcare).

In vitro activity assay of ClyR

E. coli Nissle 1917 transferred with plasmid A and B, Lactobacillus casei subsp. casei (ATCC334)

1) The culture of Lactobacillus casei subsp. casei (ATCC334)

2) In vitro assay

The value of OD600 was monitored using Multiscan Spectrum (BioTek). Table 1. The protein concentration of the culture supernatant of E. coli with plasmids A and B.

..

Proof of function

As can be seen from figure 4, 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 4.

Figure 4. The SDS-PAGE of supernatant..

In vitro activity assay of ClyR

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 5).

Figure 5. The in vitro assay of ClyR...

Improvement by 2022 SHSID

Compared to the old part projects BBa_K4030009，which is a biological part submitted by iGEM21_Shanghai_United_HS in 2021, the ClyR is under araBAD promoter, which is induced by arabinose. And ClyR is linked with ompA and this sequence is inserted into plasmid. The constructed plasmid exhibited weak activity. In this part, we selected the dextranase DexA. DexA can inhibit the formation of biofilm and destroy the pre formed biofilm dextranase DexA in vitro. We designed a genetically engineered bacterium that produces a glucanase DexA and a phage lyase ClyR. The former can effectively prevent the formation of biofilm and dissolve the formed biofilm, while the latter can specifically kill Streptococcus mutans. The genetically engineered bacteria prepared in this project have great potential in the prevention and treatment of dental caries.

J23101-DexA70-rrnBT1-T7TE

Profile

Name: J23101-DexA70-rrnBT1-T7TE

Base Pairs: 2148 bp

Origin: Streptococcus, synthetic

Properties: DexA70 is placed the downstream of J23101 for the expression.

Usage and Biology

BBa_K4280004 is the construct of J23101-DexA70-rrnBT1-T7TE. Dental caries or tooth decay is a common disease, which not only directly affects human oral health, but also often causes adverse symptoms in other parts of the body [1]. According to the global disease statistics in 2016, the incidence rate of dental caries in the population is second only to upper respiratory tract infection, ranking second among common disease. Dental plaque is the primary condition and initiating factor of dental caries. Research shows that the formation of dental plaque is the result of the joint action of many kinds of bacteria. Oral microorganisms based on high-throughput sequencing showed that Streptococcus mutans, lactobacillus, actinomycetes, etc. in plaque were closely related to the occurrence of caries [2-3]. They metabolize and produce various acids, causing the destruction of organic and inorganic substances in teeth to form visible cavities. Effective removal or inhibition of dental plaque formation has become an important means to prevent dental caries [4]. Brushing teeth and flossing are the most basic and practical methods to remove dental plaque, but it is difficult for many people to master and adhere to them. On the other hand, chemical agents such as chlorhexidine or triclosan, which have been used clinically, have significant cytotoxicity in vitro, as well as side effects such as tooth staining. Although fluoride can prevent dental caries by enhancing the acid resistance of teeth, it is difficult to ensure the stability of fluoride concentration. In this sense, the demand for new antibacterial substances from other sources is increasing. Phage lyase is a kind of glycoside hydrolase encoded by phage, which has the function of digesting the cell wall of bacterial host cell. When phage lyase acts on gram-negative bacteria, it shows high bactericidal activity, species specificity and safety in vitro and in vivo, so it has broad application prospects [5]. Biofilm is a highly organized and structured bacterial cell community hidden in the extracellular matrix. Most biofilm substrates are polysaccharides, and dental biofilms are no exception. (1-3) and (1-6) -α-D-glucan polysaccharides are the main components of Streptococcus mutans biofilm matrix. The biofilm produced by Streptococcus mutans is the main obstacle to the effect of various antibacterial agents. The best treatment must be able to eradicate biofilms. Glucanase can inhibit biofilm by hydrolyzing α-1,6-glycosidic bond. Studies have shown that dextranase DexA can inhibit the formation of biofilm and destroy the pre formed biofilm in vitro [6-7].

Figure 1. The schamatic uasge of smDexA for debating dental caries.

Construct design

1. Construction of the J23101-DexA70-rrnBT1-T7TE

The schame of the construction of J23101-DexA70-rrnBT1-T7TE was shown as figure

Figure 2. Construction schame of J23101-DexA70-rrnBT1-T7TE.

The profiles of every basic part are as follows:

BBa_K4280000

Name: J23101

Base Pairs: 35 bp

Origin: E.coli, genome

Properties: a strong constitutive promotor.

Usage and Biology

BBa_K4280000 is a commonly used strong constitutive promotor in bacteria.

BBa_K4280001

Name: dexA70

Base Pairs: 1899 bp

Origin: Streptococcus, genome

Properties: a Sec-dep a phage lyase with glycoside hydrolase activity acting against bacteria.

Usage and Biology

BBa_K4280001 is the coding sequence of DexA70, and it could inhibit the formation of biofilm and destroy the pre formed biofilm in vitro.

BBa_K4280002

Name: ompA signal peptide

Base Pairs: 63 bp

Origin:E.coli, genome

Properties: encoding the signal peptide of the OmpA protein.

Usage and Biology

BBa_K4280002 is the signal peptide of the OmpA protein, and it has been used for the secretion of proteins downstream.

BBa_K4280003

Name: rrnBT1-T7TE

Base Pairs: 87 bp

Origin:E.coli, genome

Properties: a transcriptional terminator.

Usage and Biology

BBa_K4280003 is the commonly used transcriptional terminators in the expression of heteroglous proteins in the engineering E. coli strains.

Experimental approach

1. PCR amplification.

For the construction, we firstly amplified DexA and pUC57 fragments by PCR assay. As indicated in Figure 3, DexA and pUC57 strands were correctly replicated.

Figure 3. DexA and pUC57 strands are correctly replicated.

Identification of DexA (1899 bp) and pUC57 (2126 bp) DNA strands by agarose gel electrophoresis.

2. Enzymatic digestion verification.

Secondly, we used double enzyme digestion to construct puc57-kan-mini-J23101-OmpA-smDexA-TT plasmid. PCR was used to verify the monoclonal colony of the strain. As indicated in Figure 2, 1 and 2 were positive clones.

Figure 4. pUC57-kan-mini-J23101-OmpA-smDexA-TT are correctly replicated.

Identification of puc57-kan-mini-J23101-OmpA-smDexA-TT by agarose gel electrophoresis.

3. Sequning of constructed puc57-kan-mini-J23101-OmpA-smDexA-Ttplasmid.

Thirdly, gene sequencing is used to double verification. As indicated in Figure 5, puc57-kan-mini-J23101-OmpA-smDexA-TT sequence was correct.

Figure 5. Sequencing of J23101-DexA70-rrnBT1-T7TE.

4. SDS-PAGE analysis of the target protein.

Fourthly, J23101-OmpA-smDexA-TT was purified according to the protocol. Then we used SDS-PAGE to test the purity of puc57-kan-mini-J23101-OmpA-smDexA-TT. As shown in Figure 6, the puc57-kan-mini-J23101-OmpA-smDexA-TT was purified successfully.

Figure 6. SDS-PAGE assay.

Glue diagram of DexA and ClyR proteins. MS is medium supernatant, P is precipitation, and S is supernatant. The molecular weight of DexA protein is 74.33 kDa. The molecular weight of ClyR protein was 30.49 kDa.

5. Activity assassy.

Finally, we test the function of the strain. As indicated in Figure 7, The plasmid we constructed had a good bactericidal effect.

Figure 7. Strain functional test (A) The white circle of ClyR, dexA-6, ClyR + DexA-6, ClyR + DexA-1 and DexA-1 covers the entire plate, adding up to 6.8 cm. (B) The white circle of ClyR is 4.2 cm; The white circle of DexA-6 is 1.4 cm; The white circle of ClyR + DexA-6 is 1.3 cm; The white circle of ClyR + DexA-1 is 1.6 cm; The white circle of DexA-1 is 1.0 cm..

Reference

1.Yang, H. et al. A chimeolysin with extended-spectrum streptococcal host range found by an induced lysis-based rapid screening method. Sci. Rep. 5, 17257; doi: 10.1038/srep17257 (2015).

2.McGowan S, Buckle AM, Mitchell MS, Hoopes JT, Gallagher DT, Heselpoth RD, Shen Y, Reboul CF, Law RH, Fischetti VA, Whisstock JC, Nelson DC. X-ray crystal structure of the streptococcal specific phage lysin PlyC. Proc Natl Acad Sci U S A. 2012 Jul 31;109(31):12752-7. doi: 10.1073/pnas.1208424109.

3. Seo EJ, Weibel S, Wehkamp J, Oelschlaeger TA. Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins. Int J Med Microbiol. 2012 Nov;302(6):276-87. doi: 10.1016/j.ijmm.2012.05.002.

4. Liu N, Li X, Wang M, Zhang F, Wang C, Zhang K, Wang H, Xu S, Hu W, Gu L. DexA70, the Truncated Form of a Self-Produced Dextranase, Effectively Disrupts Streptococcus mutans Biofilm. Front Microbiol. 2021 Sep 28;12:737458. doi: 10.3389/fmicb.2021.737458.

5. Ogawa A, Furukawa S, Fujita S, Mitobe J, Kawarai T, Narisawa N, Sekizuka T, Kuroda M, Ochiai K, Ogihara H, Kosono S, Yoneda S, Watanabe H, Morinaga Y, Uematsu H, Senpuku H. Inhibition of Streptococcus mutans biofilm formation by Streptococcus salivarius FruA. Appl Environ Microbiol. 2011 Mar;77(5):1572-80. doi: 10.1128/AEM.02066-10.

6. Liu N, Li X, Wang M, Zhang F, Wang C, Zhang K, Wang H, Xu S, Hu W, Gu L. DexA70, the Truncated Form of a Self-Produced Dextranase, Effectively Disrupts Streptococcus mutans Biofilm. Front Microbiol. 2021 Sep 28;12:737458. doi: 10.3389/fmicb.2021.737458.

7. Zayed SM, Aboulwafa MM, Hashem AM, Saleh SE. Biofilm formation by Streptococcus mutans and its inhibition by green tea extracts. AMB Express. 2021 May 25;11(1):73. doi: 10.1186/s13568-021-01232-6. PMID: 34032940; PMCID: PMC8149520.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 113
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 113
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 113
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 113
  
• 1000
  COMPATIBLE WITH RFC[1000]