Part:BBa_K4002005

endo-pgaA

endo-pgaA

BBa_K4002005

Name: endo-pgaA

Base Pairs: 1113bp

Origin: Aspergillus niger SC323, genome

Properties: An enzyme degradation of pectin

Usage and Biology

Polygalacturonase is an enzyme that hydrolyzes the alpha-1,4 glycosidic bonds between galacturonic acid residues. It is also known as pectin depolymerase, PG, pectolase, pectin hydrolase, and poly-alpha-1,4-galacturonide glycanohydrolase. This part as a repair template DNA was connected with homology arm of HXK1.

Experimental approach

Construction of repair template The repair template DNA containing PgaA gene (Fig. 3A) was generated by the overlap-PCR method. We determined the concentration of PgaA from recombinant S. cerevisiae strains. Samples from the culture media, total cell lysates and the soluble portion of cell lysates were collected and subjected to DNS colorimetric assay.

Table 1. Measurement of PgaA concentration and unit of activity in various samples.

As shown in Table. 1, the concentration of PgaA in the culture media of sample -1 and -2 were determined at about 0.034 mg/ml and 0.028 mg/ml, respectively, which were relatively higher than that of cell lysates (0.009 mg/ml and 0.007 mg/ml), suggesting that most of the PgaA proteins were secreted into the culture media. In addition, in the cell lysates of sample 1, we detected ~76% of PgaA in the soluble supernatants, implying that most of the PgaA in cells are soluble.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 431
  Illegal BglII site found at 989
  Illegal XhoI site found at 949
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 688
  Illegal BsaI.rc site found at 190
  Illegal BsaI.rc site found at 1060