Part:BBa_K4280006
puc57-kan-mini-J23101-OmpA-smDexA-TT
puc57-kan-mini-J23101-OmpA-smDexA-TT
Profile
Name: puc57-kan-mini-J23101-OmpA-smDexA-TT
Base Pairs: 4033 bp
Origin: Streptococcus, synthetic
Properties: smDexA is placed the downstream of OmpA for the expression.
Usage and Biology
BBa_K4280006 is the construct of puc57-kan-mini-J23101-OmpA-smDexA-TT. 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].
Construct design
1.Construction of the puc57-kan-mini-J23101-OmpA-smDexA-TT
The schame of the construction of puc57-kan-mini-J23101-OmpA-smDexA-TT was shown as figure 2.
The profiles of every basic part are as follows:
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_K4280005
Name: pUC57-Kana-min-backbone
Base Pairs: 2126 bp
Origin: E.coli, synthetic
Properties: a cloning vector commonly used.
Usage and Biology
BBa_ K4280005 is the backbone of the cloning vector pUC57 with resistence to kanamycin.
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.
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.
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.
4. SDS-PAGE analysis of ClyR
Glue diagram of ClyR proteins. MS is medium supernatant, P is precipitation, and S is supernatant. he molecular weight of DexA protein is 74.33 kDa. As can been from figure 6, DexA was successfully expressed, which set important stage for future engineering.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1186
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