Part:BBa_K5520004

pT7-LacO-His-CDA

This part contains BBa_K2406020, BBa_B0034, BBa_K5520002 and BBa_K731721. BBa_B0034 contains a natural ribosome binding site (RBS) to facilitate the expression of downstream genes. By inducing efficient expression of the CDA protein through the addition of IPTG, the protein can subsequently be purified using the His tag for further enzyme activity tests and product polymerization.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 149
  Illegal NheI site found at 897
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 182
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 641
  

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. Using CDA-F and CDA-R as primers, we amplified PET-28a-CDA as a vector using Gibson assembly. The T7 promoter、RBS、 lac operator, CDA gene, 6×His tag, kana antibiotic gene, and T7 terminator on the vector PET-28a were already inserted before experiments.

Detection of PET-28a-CDA

1. SDS-PAGE analysis of CDA protein

We utilized SDS-PAGE to detect the expression of CDA and obtained the picture below, which indicates successful expression verification. The theoretical size of CDA protein is 31.5kDa.

2. Enzymatic activity determination of CDA

The enzymatic activity of CDA was assessed using a chromogenic substrate assay. In this method, the substrate p-nitroacetanilide was hydrolyzed by CDA to produce p-nitroaniline, which exhibited a characteristic absorbance at 400 nm. Figure below presents the standard curve for p-nitroaniline. The CDA enzyme activity was calculated using the formula provided in DESIGN 3.1, yielding an activity of 51.56 U/mL.

3. Analysis of the degree of deacetylation of the CDA hydrolysis product

The enzyme activity of CDA was performed by chromogenic substrate method which is a common way to quantify amidase activity. We selected p-nitroacetanilide as the substrate, which can be hydrolyzed to p-nitroaniline. The concentration of p-nitroaniline can be measured by the characteristic absorption at 400 nm. Chitin deacetylase (CDA) activity unit is defined as the amount of enzyme required to produce 1 μg of p-nitroaniline per hour is defined as one unit of enzyme activity. Then we obtained CDA Enzyme Activity by using the enzyme activity formula (1):

A400：OD400 of experiment groups, A0：OD400 of blank group, N: dilution factor, K: slope value of p-nitroaniline standard curve, T: reaction time (h), The product chitosan was obtained with a deacetylation degree of 65.24% while 1% colloidal chitin was hydrolyzed by CDA.