Part:BBa_K4450007

UDP-acetylglucosamine pyrophosphorylase

UDP-N-acetylglucosamine pyrophosphorylase cluster

Profile

Name: UDP-N-acetylglucosamine pyrophosphorylase
Base Pairs: 1495 bp
Origin: Saccharomyces Cerevisiae ATCC 201388
Properties: Complex of enzymes responsible for activation of acetylglucosamine with UTP

Usage and Biology

Complex of UDP-N-acetylglucosamine pyrophosphorylase (EC 2.7.7.23) is the last enzyme in the hexosamine biosynthesis pathway, responsible for adding UDP to form the final product, UDP-acetylglucosamine. The enzyme utilizes N-acetylglucosamine-1-phosphate and UTP as substrates [1].

Figure 1: Vector map of gene cluster in pUC19 plasmid.

The complex was inserted in pUC19, and transformed into Escherichia coli dh5α using heat-shock transformation. This complex was used to troubleshoot our cloning process, through protein expression and colorimetric assays.

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 1396
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal SpeI site found at 1396
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 413
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 1396
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 1396
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1319
  

Characterization

Initial isolation of UAP1 from Saccharomyces cerevisiae s288c using primers found under the design tab in Part: BBa_K4450002 for single-gene assembly. From figure 2, it is possible to see a strong band from PCR amplification. This gene was then successfully purified (using gel extraction).

Figure 2: Gel electrophoresis of amplified gDNA from S. cerevisiae using primer pairs for single-gene isolation found in part: BBa_K4450002.

The UAP1-pUC19 plasmid was cloned into competent E. Coli. By restriction digestion (picture not shown), we were not able to confirm successful insert of UAP1 complex into vector. Either way, we did run an SDS-page with crude lysate from the UAP1-pUC19 mutant and compare it to a E. Coli with an empty pUC19. Figure 3 show a comparison between this mutants profile and non-mutant profile, with a band showing around 53.5 kDa in the mutant, which is where we expect the UDP-N-acetylglucosamine pyrophosphorylase enzyme to be.

Figure 3: SDS-page of 4 colonies UAP1-pUC19 (right) and empty pUC19 (left).

To further validate successful cloning, we ran a simple colorimetric phosphorylation assay using Biomol Green reagent to quantify amount of phosphate in lysate [2]. Figure 4 shows the plate after incubation with crude lysate. Here it is apparent that something is happening because of the addition of lysate due to the darker color compared to the blank measurement.

Figure 4: Image of colorimetric phosphorylation assay of 4 colonies E. coli transformed with UAP1.

Below, in figure 5, the absolute measurements of the assay at A620 after incubation is determined. The samples were prepared in triplicates from lysate prepared from different colonies, and compared to the unmodified parental cell with an empty pUC19 vector. From this, we can see that it is a significant different between the empty pUC19 parental cell, and the modified UAP1-pUC19 cells. This suggests, that the enzyme is present.

Figure 5: Absorbance reading of phosphorylation assay of 4 colonies E. coli transformed with UAP1.

References

[1] Mio, Yabe, T., Arisawa, M., & Yamada-Okabe, H. (1998). The eukaryotic UDP-N-acetylglucosamine pyrophosphorylases: Gene cloning, protein expression, and catalytic mechanism. The Journal of Biological Chemistry, 273(23), 14392–14397. https://doi.org/10.1074/jbc.273.23.14392

[2] Fang, Du, T., Raimi, O. G., Hurtado‐Guerrero, R., Urbaniak, M. D., Ibrahim, A. F. M., Ferguson, M. A. J., Jin, C., & Aalten, D. M. F. (2013). Genetic and structural validation of Aspergillus fumigatus UDP‐N‐acetylglucosamine pyrophosphorylase as an antifungal target. Molecular Microbiology, 89(3), 479–493. https://doi.org/10.1111/mmi.12290