Difference between revisions of "Part:BBa K4137009"

Latest revision as of 08:13, 10 October 2022

T7+RBS+YebF-GS Linker-chi18h8-6xHis+B1006

This part synthesizes and secretes a novel bacterial chitinase Chi18H8, which can be released extracellularly through e.coli Sec pathway (SecB-SecA-SecYEG, OmpF). This system continuously promotes the overexpression of Chi18H8 to lyse the chitin cell wall of the fungus fusarium oxysporum f.sp cubense, inhibiting fungal proliferation.

Fig.1 Chitinase (chi18h8) SEC secretion construct.

Construct Designs

We attached a 6x His-Tag downstream of the chi18h8 sequence for purification purposes. Attached to the upstream of the chi18h8 sequence is a glycine-serine linker (GS linker) (BBa_J18921) that links chi18h8 to a yebF secretion tag (BBa_K2922002), which forms our open reading frame (ORF). We used a T7 strong promoter and flanked RBS (AAGGAG) upstream of the ORF. The terminator BBa_B1006 is attached downstream of the ORF.

Characterization

Chi18H8 expression was performed in 50 ml of expression medium with BL21(DE3) E. coli. We closely followed Berini et al.’s protocol because Chi18H8 required specific expression protocols. We performed expression at two temperatures, 37˚C and 30˚C; the former was used as a reference for standard in vitro settings, and the latter was used to simulate soil temperature. 5 ml of expression medium was used for cell harvest at time intervals of 2h and 4h. We heated the mixture at 95˚C for 10 minutes, centrifuging at 4˚C, 16000g to extract the supernatant. An additional 95˚C heating and 100 µl PBS were added to eliminate clumping of pellets.

Fig.2 20 µl of extracted Chitinase (chi18h8) under SDS-PAGE analysis.

Due to excessive protein quantity and dyeing, the protein bands for Chi18H8 were obscured but are still distinguishable.

Sequence and Features