Part:BBa_K5520005

pT7-LacO-His-CsnB

This part is composed of BBa_K2406020, BBa_B0034, BBa_K5520003 and BBa_K731721. We used T7 promoter and RBS to control the expression of CsnB. 6×His tag was utilized for protein isolation and purification. lac operator was inserted to regulate the utilization of lactose by bacteria.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 844
  Illegal NotI site found at 804
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 813
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 153
  Illegal AgeI site found at 636
  
• 1000
  COMPATIBLE WITH RFC[1000]

Plasmid construction

PET-28a linearized vector (5371 bp) was amplified using PET-28a plasmid from the laboratory conserved as template and ZT-F and ZT-R as primers. Marine Bacterium Bacilius SP. BY01 strain genome was used as template, CsnB-F and CsnB-R were used as primers, and CsnB gene fragment (741 bp) was amplified. Gibson assembly was used to ligate the CsnB gene fragment to the PET-28a linearized vector. Colony PCR (928 bp) was performed on the transformed colonies with primers CSNB-CX-F and CSNB-CX-R. Positive colonies with correct colony PCR were transfected, plasmid was extracted, and the recombinant plasmid was obtained after sequencing and verification: PET-28a-CsnB.

Detection of PET-28a-CsnB

1. 1. SDS-PAGE analysis of CsnB protein

We employed SDS-PAGE to assess the expression level of CDA, and the resulting image is provided below. This confirms the successful verification of CDA expression. The theoretical size of the CsnB protein is 31.7 kDa.7 BBa_K5520006

2. 2. Product Analysis of CsnB

The hydrolysis of soluble sugars from chitosan by CsnB was investigated using thin-layer chromatography (TLC). As depicted in Figure 5, the hydrolysis process was found to be incomplete at both 30 minutes and 150 minutes, with the predominant products being (GlcN)3 and (GlcN)4. After 13 hours, the hydrolyzed products predominantly consisted of (GlcN)2 and (GlcN)3. No GlcN was detected after 17 hours, suggesting that CsnB is an endo-chitanase.