Team:ZJU-China/CompositePart

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COMPOSITE PARTS  
OVERVIEW  



The injury detector we made is based on a cell-free device with three connected enzymes. HRP(Horseradish Peroxidase) is a commonly used enzyme as clinical testing reagent which catalyzes the reductive cleavage of hydrogen peroxide by an electron donor. As one of the three enzymes for diagnosis, it is of vital importance.


Meanwhile, we found that SpyTag forms a spontaneous isopeptide bond to SpyCatcher, and SdyTag/SdyCatcher, SnoopTag/SnoopCatcher are the same[1][2][3]. The tag/catcher system is a useful tool to link HRP with other protein via covalent bond.




Design

This part consists of BBa_J64997, BBa_B0034, BBa_B0010, BBa_B0012(previous parts) and BBa_K2721029(new part) which codes for HRP, SnoopTag and SdyCatcher. Flanked by SnoopTag and SdyCatcher, HRP can be connected with the other two enzymes to form multienzyme complex. We obtain the original DNA sequence of HRP, SnoopTag and SdyCatcehr from the references below[2][3][4]. After optimizing and synthesizing, we cloned the target sequence into vector PET-26b(+) and successfully detected the production.


Result

We utilized SDS-PAGE and Western blot to detect the expression of SnoopTag-HRP-SdyCatcher. The primary antibody is Mouse-anti-His mAb.

Fig. 1 SdyCatcher-HRP-SnoopTag| (A) SDS-PAGE, Lane M1 is protein marker, Lane 1 is BSA (2.00 µg), Lane 2 is SdyCatcher-HRP-SnoopTag. (B) Western Blot, Lane M2 is protein marker, Lane 3 is SdyCatcher-HRP-SnoopTag.

Besides, we used BSA Standard Curve to qualifity the expression of SdyCatcher-HRP-SnoopTag.

Fig. 2 Standard curve for Bradford assay

Sample

OD595

Concentration

SdyCatcher-HRP-SnoopTag

0.398

0.80mg/ml
Table. 1 qualification result of three complex.

References

[1]Zakeri (B) Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A. 2012 Mar 20;109(12):E690-7.

[2]Veggiani G, Nakamura T, Brenner MD, Gayet RV, Yan J, Robinson CV, Howarth M.Programmable polyproteams built using twin peptide superglues. Proc Natl Acad Sci U S A. 2016 Feb 2;113(5):1202-7.

[3]Tan LL, Hoon SS, Wong FT. Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly. PLoS ONE 11(10): e0165074.

[4]Edwards,R. SYNTHETIC GENE. British Bio-Technology Ltd.

[5]P Hengen. Purification of His-Tag fusion proteins from Escherichia coli. Trends in Biochemical Sciences, 1995,20(7): 285-286.