Difference between revisions of "Part:BBa K3792007"

Latest revision as of 01:07, 20 October 2021

NodCIJ Generator II

The NodCIJ Generator with a medium promoter does not produce a lot of the NodC protein, NodI protein, and NodJ protein. The NodC protein produces chitin and NodI and NodJ transports nodulation factors.

This part consists of 2566 base-pair length. It includes a promoter, 3 ribosome binding sites, 3 protein-coding sequences, and a terminator. We used promoter K2832161, a weak promoter from the BIOFAB collection. In addition, we also used protein-coding sequences from Chitin synthase from Rhiozobium leguminosarum, Nodl a protein possibly involved in transporting chitin across the cell membrane, and NodJ, a protein also possibly involved in the transportation of chitin

This generator was assembled into the pSB1K3 plasmid, and then transformed into E.Coli NEB 5-Alpha. To test if the cells were secreting chitin, The FSU 2021 team used the Chitosan Assay Kit (XAN-5126) available from Cell Biolabs. The manual for this kit contains the protocols for converting chitin to chitosan, along with a protocol for detecting chitosan.

Chitin from the cells was converted into chitosan by reacting with 12.5M NaOH at 95°C for 24 hours. The solution was then neutralized with acetic acid. The assay was then performed on the supernatant of the cell culture. Sequence and Features