Part:BBa_K4030001

arab

Profile

Name: DexA70

Base Pairs: 1899 bp

Origin: Streptococcus, genome

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

Usage and Biology

BBa_K4030001 is the coding sequence of DexA70. 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 ClyR for debating dental caries..

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.

Usage and Biology

BBa_K4030001 is a coding sequence of arab, which is an enzyme used to hydrolyze arabinose from arabinoxylans, a carbohydrate that appears normally in foods.

Experimental approach

Group 1

Plasmid puc57-kan-mini-J23101-OmpA-araB-TT (Plasmid A) was transformed to E. coli Nissle 1917 by electroporation. We tried to monitor the concentration of the reducing sugar arabinose and thus access the activity of AraB.

The concentration of reducing sugar was determined by the DNS kit and the A540 were recorded accordingly.

..

Group 2

Plasmid A and plasmid pBAD-Myc-HisA-OmpA-amilGFP (Plasmid C) were co-transformed to E. coli Nissle 1917 by electroporation. The existence of AmilGFP was monitored by the fluoroscopic examination using SpectraMax i3x..Read the fluoroscopic data of the supernatant, and recorded the average data. After this timepoint, 100 ul of the culture was sampled and marked as “6 h”, “8 h”, “10 h”, “12 h” and “13 h”, respectively, and the fluoroscopic data were recorded accordingly.

Group 3

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.

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

Proof of function

Figure 1. The concentration assay of the reducing sugar in bacteria transformed with plasmid A..

All the data at “0 h” with different concentration of araboxylan was referred as blank, and the concentration of the reducing sugar could be calculated.

As can be seen from figure 1, in the transformed bacteria with plasmid A, the concentration of the reducing sugar increased with the elongation of the incubation time. The plasmid A can work normally and possess hydrolyzing arabinoxylan activity, which supply the arabinose for the expression of ClyR.

AmilGFP expression in cells transformed with plasmid A

Figure 2. The fluoroscopic data of cells transformed with plasmid A..

In the cells transformed with only plasmid A, the fluoroscopic data of cells with the addition of different concentration of araboxylan showed no difference with that of cells without the addition of araboxylan. This data suggested no AmilGFP was produced in cells with or without addition of araboxylan.

AmilGFP expression in cells transformed with plasmids A and C

Figure 3. The fluoroscopic data of cells transformed with plasmids A and C..

In the cells transformed with plasmids 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.

AmilGFP was successfully expressed, suggesting the feasibility and possibility of the inducible secretory expression of ClyR.

..

Figure 4. The SDS-PAGE of supernatant. ..

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. 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.

In the engineering cells containing plasmid A and plasmid B, the expression of ClyR was successful.

References

5,Pitts, N. B., Zero, D. T., Marsh, P. D., Ekstrand, K., Weintraub, J. A., Ramos-Gomez, F., Tagami, J., Twetman, S., Tsakos, G., & Ismail, A. (2017). Dental caries. Nature Reviews Disease Primers, 3(1). https://doi.org/10.1038/nrdp.2017.30

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 633
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 633
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 633
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 633
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 633
  
• 1000
  COMPATIBLE WITH RFC[1000]