Part:BBa_K2380001

Chitin synthase NodC with RBS and Anderson-Promoter

The Chitin Synthase (CHS) NodC from Rhizobium leguminosarum is a N-acetylglucosaminyl transferase, that catalyzes the formation of chitin oligomers by using UDP-acetylglucosamine as donor molecule and N-acetylglucosamine as acceptor. The expression of NodC is regulated by the constitutive Anderson-promoter system (BBa_K2380025). For further characterizations please visit BBa_K2380000 or our [http://2017.igem.org/Team:TU_Darmstadt/project/chitin_synthase wiki].

Plasmidcard

Characterization

Calcofluor white is a stain that fluoresces blue when in the presence of chitin and UV light. As seen below, colonies with that contain a gene expressing chitin (left) fluoresce blue much brighter than the wildtype strain. The calcofluor white assay was used to determine if chitin was being produced by our cells. Both a blue and a white colony were selected from the pGEM plate, and it was assumed that the blue colony would not have the gene inserted and the white colony would. Each colony was suspended in 200 microliters of PBS, and then 20 microliters of calcofluor white was added to the tube. The cells then sat for one minute, and then 40 microliters were pipetted onto a microscope slide. Once the glass coverslip was put on, the slide was analyzed under a fluorescent microscope. The cells were observed on a normal light setting and with UV light, and then an overlay image was created to compare those two views.

The figure shown below shows NodC with an Anderson on the top and a negative control from the same plate on the bottom. The chitin product was shown to be produced since the cells fluoresce after being mixed with the calcofluor white assay. The control was used as a comparison to show how wildtype E. coli does not have most of its cells fluoresce.

https://2019.igem.org/File:T--Purdue--G1comparison.jpg

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 36
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 438
  
• 1000
  COMPATIBLE WITH RFC[1000]