Part:BBa_K4768004

Recombinant Pyrimidine/purine nucleoside phosphorylase (ppnP) from E. coli

Biosensing inducible system to express E.coli nucleoside phosphorylase (ppnP) in PURE system

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Introduction

Figure 1: Pyrimidine/purine nucleoside phosphorylase ppnP part

The part (BBa_K4768004) includes the E. coli Pyrimidine/purine nucleoside phosphorylase gene, controlled by a T7 promoter with an operator sequence called dhdO. This part is very important in our project because the encoded enzyme is responsible for the conversion of Tegafur (a prodrug) into 5-Fluorouracil (an active drug).

Construction

The ppnP gene was obtained from Dr. Baixing Wu, an Associate Professor at the RNA Biomedical Institute, Medical Research Center at Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University. Dr. Wu is the author of the article titled "Crystal structures of a new class of pyrimidine/purine nucleoside phosphorylase revealed a Cupin fold" [1]. The ppnP gene was placed under the control of a T7 promoter with an operator site known as dhdO. The synthesis of the gBlock of this part was carried out and provided by IDT (Integrated DNA Technologies).

We amplified the gBlock by PCR using the following primers(from 5' to 3'):

T7-term-F : AGTTCCTCCTTTCAGCAAAAAACCCCTCAAGACC

T7-prom-R : GAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGG

Figure 2: Figure 2: Migration of ppnP PCR products into a 0.8% agarose gel and EtBr staining.

Characterisation

We attempted to produce E. coli purine/pyrimidine nucleoside phosphorylase (Ppnp) in two different PURE system kits with different protein folding properties (PUREfrex2.0 and PUREfrex2.1). . Tegafur at a final concentration of 119 µM and the gene encoding Ppnp under T7 polymerase control were added in each sample. Tegafur and 5-FU concentrations were analyzed by HPLC at time zero and after 12 hours incubation at 37°C (Table 1).

Table 1: Table 1: Estimated concentrations of Tegafur and 5-FU at time zero (T-0h) and after 12 hours (T-12h) of incubation at 37°C under different conditions (as indicated above).

Conclusion and Perspectives

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References

1. article 1 xxxxxxxx
2. article 2 xxxxxxx