Part:BBa_K3286106

Shorter version of PD1764

This protein is a shorter (sh) version of BBa_K3286105. Due to isolation troubles, the first 20 amino acids have been removed from this protein. The part contains a chitin-binding domain that is essential for chitin binding capacities [1].

[1] F. Labroussaa, A. R. Zeilinger, and R. P. P. Almeida, “Blocking the Transmission of a Noncirculative Vector-Borne Plant Pathogenic Bacterium,” Mol. Plant-Microbe Interact., vol. 29, no. 7, pp. 535–544, Jul. 2016.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 787
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 294
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Characterization

Figure 1: Percentage of bound protein to chitin ((B - A )/ B). 55% of PD1764sh has bound to chitin in this assay. The influence of chitin on protein concentration in general is negligible, as the BSA sample indicates. A= "protein concentration with chitin in ug/ml" and B= protein concentration without chitin in ug/ml".

To prove chitin binding, a chitin binding assay was conducted. In a 2 ml microfuge tube, 500 mg of chitin solution (10 mg/ml) was added, together with 50 ug of protein in solution. The final volume was adjusted, using MQ, to 500 ul. Then, the tubes were incubated at room temperature in a shaking block at 600 rpm. After 1 hour, the tubes were centrifuged for 3 minutes at 13000 x g. Then, the supernatant was used to perform a Bradford assay with.

In this experiment, 4 different samples were used (triplicates): PD1764sh with chitin, PD1764sh, BSA and BSA with chitin. The difference in protein concentration was calculated, which was divided by the protein samples without chitin. The two results were plotted in Figure 1.

Chitin binding could potentially increase with longer incubation or longer centrifugation.