Difference between revisions of "Part:BBa K3792006"

Revision as of 02:43, 22 October 2021

NodCIJ Generator I

The NodCIJ Generator I has a BIOFAB promoter with a strength recorded at x fluorescence per cell. The strongest promoter in the collection has a strength of y . NodC is a chitin synthase that produces tetrameric chitooligomers. NodI and NodJ are transport proteins that usually secrete.

This part consists of 2555 base-pair length. It includes a promoter, 3 ribosome binding sites, 3 protein-coding sequences, and a terminator. We used promoter K2832184, 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.

This data reflects the absorbance of chitosan from the assay kit. From our data, we can see how the supernatant of the chitin secretion cells had an absorbance of 2.477. However, the LB medium had a very high absorbance of 1.515. As a result, we suspect that our medium, LB, has chitosan. We can confirm there is chitosan in our supernatant, however, we cannot be sure if this is directly due to the cells or due to the medium LB. We hypothesize that there is chitosan in both our cells and the LB medium, as a result, we will further be testing our chitin secretion cells in a middle medium to confirm if our cells directly secret chitosan.

Sequence and Features