Difference between revisions of "Part:BBa K4030009"

Revision as of 02:39, 20 October 2021 (view source) Hrr1225 (Talk | contribs) ← Older edit		Revision as of 09:36, 20 October 2021 (view source) SNX (Talk | contribs) (→‎Base Pairs: 4837bp) Newer edit →
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	== Profile ==		== Profile ==
	==== Name: Myc-HisA-OmpA-ClyR-6His-TT ====		==== Name: Myc-HisA-OmpA-ClyR-6His-TT ====
−	==== Base Pairs: 4837bp ====	+	==== Base Pairs: 1007bp ====
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	==== Origin: E. coli , Streptococcal phage, synthetic ====		==== Origin: E. coli , Streptococcal phage, synthetic ====
	==== Properties: Inducible ClyR expression system to resit Dental caries ====		==== Properties: Inducible ClyR expression system to resit Dental caries ====

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Revision as of 09:36, 20 October 2021

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

Profile

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

Base Pairs: 1007bp

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