Difference between revisions of "Part:BBa K4137009"
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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. | 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. | ||
| − | [[File:chi-sds.png|800px|thumb|center|Fig. | + | [[File:chi-sds.png|800px|thumb|center|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. | Due to excessive protein quantity and dyeing, the protein bands for Chi18H8 were obscured but are still distinguishable. | ||
Revision as of 05:17, 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.
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.
Due to excessive protein quantity and dyeing, the protein bands for Chi18H8 were obscured but are still distinguishable.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1523
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1036
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 583
- 1000COMPATIBLE WITH RFC[1000]