Coding

Part:BBa_K4030001

Designed by: Xiaoyan Wei   Group: iGEM21_Shanghai_United_HS   (2021-10-12)
Revision as of 01:12, 20 October 2021 by Hrr1225 (Talk | contribs)


arab

Profile

Name: arab

Base Pairs: 1479bp

Origin: Bacillus pumilus

Properties: Secreting arabinosidase to hydrolyze polysaccharides into monosaccharides.

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 3 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 2 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

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


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]


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