Part:BBa_K3789010

PNP enzyme secretion system

This composite part consists of pGK1 promoter, Mating factor alpha prepro-leader (MF alpha), 6 x glycine, PNP1 coding sequence, and ADH1 terminator. The product of this system is a secretable purine nucleoside phosphorylase(PNP) together with a HA tag. MF alpha enables the enzyme to be secreted. The six glycines between MF alpha and PNP1 coding sequence avoid the interruption between them, enabling them to form correct spatial structure.

PNP1 Activity Assay

PNP1 is an enzyme that could break down the inosine to hypoxanthine, the developer could convert the hypoxanthine into urea acid. Urea acid is measured at a wavelength of OD =293nm. We add the supernatant of fermentation broth into the reaction mix, then measure the OD at 293nm every two minutes for 30 minutes, the slope of the graph will show the activity.

We add the inosine and developer into the PNP1 assay buffer at room temperature to form a reaction mix. Add the supernatant of wild type strain and PNP1 strain respectively as the test groups, at the same time, prepare exactly the same reagents except for the developer as the background group. Dividing into U.V. transparent plate(96-well). Then measure the absorbance at OD=293nm every two minutes for 30 minutes.

In the assay process, we found the slope is negative instead of positive as we predicted (Figure 1).

Then we did the standard test, it proves all the regent are effective (Figure 2).

All the test groups and background groups have a negative slope, and the gap between them is too small. We can’t detect the activity of PNP1, probably because the protein is degraded, the expression in the SC medium is insufficient.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 1448
  Illegal PstI site found at 1023
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1023
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2273
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 1448
  Illegal PstI site found at 1023
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 1448
  Illegal PstI site found at 1023
  
• 1000
  COMPATIBLE WITH RFC[1000]