Difference between revisions of "Part:BBa K4195007"

Revision as of 13:41, 10 October 2022

ttpA-his

Biology

TTPA is the phage tail tubular protein A of podophage 7. TTPA can interact with Vp0980, which acts as the receptor of TTPA on the surface of Vibrio parahaemolyticus. TTPA’s binding to Vp0980 mediates phage absorption and subsequent bacterial lysis (1).

Usage and design

A His-Tag (6×His) was added to the C-terminal of TTPA for purifying this protein. Arabinose-inducible system was used in the expression circuit at pSB1C3 then composite part BBa_K4195111 was obtained. We transformed the plasmid into E. coli SHuffle T7 to obtain the protein for further biochemical characterizations.

Characterization

Identification

When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformatants were correct. Target bands (764 bp) can be observed at the position around 750 bp (Fig. 1).

Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4195007_pSB1C3.

Purification and proof of expression

We used BBa_I0500 promoter and RBS (BBa_B0034) to express TTPA-his protein in E. coli SHuffle T7. GE AKTA Prime Plus FPLC system was implemented to get purified TTPA protein. Target bands (22.4 kDa) can be observed at the position around 20 kDa(Fig. 2).

Fig. 2 SDS-PAGE analysis of protein in lysate of E. coli BL21(DE3) and the elution samples.

Reference

1. M. Hu, H. Zhang, D. Gu, Y. Ma, X. Zhou, Identification of a novel bacterial receptor that binds tail tubular proteins and mediates phage infection of Vibrio parahaemolyticus. Emerging Microbes Infect. 9, 855-867 (2020).

Sequence and Features