Part:BBa_K2940005

Coding Sequence of AzoR with an N-terminal SpyCatcher

Coding sequence for Azoreductase AzoR with an N-terminal SpyCatcher.

Usage and Biology

This part is used for conjugation of AzoR on curli for enzyme immobilisation. The SpyCatcher is designed to be at the N-terminus of the AzoR enzyme. A 2x GGGGS flexible linker was introduced between AzoR and SpyCatcher to make sure the domains of two proteins were not interfering with each other. SpyCatcher sequence were acquired from iGEM registry (part: BBa_K1650037). And the flavin-free, oxygen insensitive azoreductase AzoR was acquired from NCBI (accession number: WP_014229193.1).

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 222
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Experimental Data

Once the gene construct was constructed, the potential improvement of enzymatic activity and substrate specificity prior conjugation to biofilm, the activity of recombinant AzoR and AzoR-SC was analyzed against two substrates, Methyl Red (MR) and Reactive Black 5 (RB5). The enzyme activity of the supernatant and cell lysate were both analyzed spectrophotometrically and plotted in the figure below. A Student’s t test was adopted to determine if uninduced samples were significantly different from its corresponding induced samples. According to the bar chart, there were azorductase activity detected in wild type BL21 samples and uninduced control samples that might be due to other oxidases or reductases produced by cells. However, they are all below 5U/mL. The enzyme activity in supernatant against two substrates for neither recombinant AzoR or AzoR-SC reached significance level, indicating the enzyme was not secreted. This corresponds to the SDS-PAGE result in the presenting study and previous study for azoreductases (Ryan, 2017). It is notable that the AzoR-SC enzyme activity in cell lysate against MR and RB5 both reached different significant level. This indicates a functional enzyme production of recombinant AzoR-SC. However, the recombinant AzoR activity in cell lysate did not reach significant against both substrates. It is clear that the produced AzoR was inactive. Besides, the activity of cell lysate AzoR-SC against MR is 700 timse higher than that against RB5. Interestingly, according to the original study that reported AzoR activity, the wild type enzyme activity against MR is 8 times of that against RB5 (Hua and Yu, 2019). These new results could mean that the specificity of AzoR against substrate MR was enhanced when linked to SpyCatcher. However, there more evidence are needed to prove this conclusion.

Reference

Hua, J.-Q. and Yu, L. (2019) ‘Cloning and characterization of a Flavin-free oxygen- insensitive azoreductase from Klebsiella oxytoca GS-4-08.’, Biotechnology letters. Ryan, A. (2017) ‘Azoreductases in drug metabolism.’, British journal of pharmacology. England, 174(14), pp. 2161–2173. doi: 10.1111/bph.13571.