Part:BBa_K4030009
Myc-HisA-OmpA-ClyR-6His-TT
Profile
Name: Myc-HisA-OmpA-ClyR-6His-TT
Base Pairs: 4837bp
Origin: E. coli , Streptococcal phage, synthetic
Properties: Inducible ClyR expression system to resit Dental caries
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.
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).
The profiles of every basic part are as follows:
BBa_K4030005
Name: ClyR
Base Pairs: 753bp
Origin: Streptococcal phage
Properties: effector in dental caries
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
Name: araBAD promoter
Base Pairs: 166bp
Origin: Escherichia coli
Properties: Inducible promoter used for protein expression
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
Name: OmpA
Base Pairs: 63bp
Origin: Escherichia coli
Properties: Outer membrane protein A
Usage and Biology
Outer membrane protein A (OmpA) is a major protein in the Escherichia coli outer membrane.
BBa_K4030003
Name: Myc
Base Pairs: 30bp
Origin: Human
Properties: epitope tag
Usage and Biology
It is an epitope tag derived from c-myc gene
BBa_K4030006
Name: 6His
Base Pairs: 18bp
Origin: synthetic
Properties: Polyhistidine tag
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.
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).
References
1,Yang, H., Linden, S. B., Wang, J., Yu, J., Nelson, D. C., & Wei, H. (2015). A chimeolysin with extended-spectrum streptococcal host range found by an induced lysis-based rapid screening method. Scientific Reports, 5(1). https://doi.org/10.1038/srep17257
2,Xu, J., Yang, H., Bi, Y., Li, W., Wei, H., & Li, Y. (2018). Activity of the Chimeric Lysin ClyR against Common Gram-Positive Oral Microbes and Its Anticaries Efficacy in Rat Models. Viruses, 10(7), 380. https://doi.org/10.3390/v10070380\
3,Selwitz, R. H., Ismail, A. I., & Pitts, N. B. (2007). Dental caries. The Lancet, 369(9555), 51–59. https://doi.org/10.1016/s0140-6736(07)60031-2
4,Seo, E., Weibel, S., Wehkamp, J., & Oelschlaeger, T. A. (2012). Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins. International Journal of Medical Microbiology, 302(6), 276–287. https://doi.org/10.1016/j.ijmm.2012.05.002
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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 113
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 113
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 113
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 113
- 1000COMPATIBLE WITH RFC[1000]
| None |