Difference between revisions of "Part:BBa K2940005"
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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. | 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. | ||
| + | <center> | ||
| + | [[Image:T--Edinburgh OG--Wendy1.JPG|500px|Figure 1]] | ||
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| + | Figure 1. Decolorization of dye by recombinant azoreductase and azoreductase- SpyCatcher. Unconcentrated crude cell lysate or supernatant were used as samples for each assay. The enzyme unit was described as unit per milliliter of sample. The error bars showed the standard error for triplicates. Wild type BL21 cell culture was used in all assays as negative control. In t test, single star indicates the induced sample reached a significance of p<0.05, triple star indicates the induced sample reached a significance of p<0.01. (A) The AzoR activity against MR in cell culture supernatant, induced and uninduced. Although AzoR-SC activity was the highest among all samples, it did not reach significant level. (B) The AzoR-SC activity against MR in crude cell lysate of cell culture, induced and uninduced. The enzyme activity of AzoR-SC reached 5000U/mL and it reached the significance of p<0.01.(C) The AzoR activity against RB5 in cell culture supernatant, induced and uninduced. The detected activity from induced samples were not significantly different form their corresponding control. (D) The AzoR-SC activity against RB5 in crude cell lysate of cell culture, induced and uninduced. The AzoR-SC activity reached the significance of p<0.05. | ||
| + | </center> | ||
==Reference== | ==Reference== | ||
Latest revision as of 18:13, 21 October 2019
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 222
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
Figure 1. Decolorization of dye by recombinant azoreductase and azoreductase- SpyCatcher. Unconcentrated crude cell lysate or supernatant were used as samples for each assay. The enzyme unit was described as unit per milliliter of sample. The error bars showed the standard error for triplicates. Wild type BL21 cell culture was used in all assays as negative control. In t test, single star indicates the induced sample reached a significance of p<0.05, triple star indicates the induced sample reached a significance of p<0.01. (A) The AzoR activity against MR in cell culture supernatant, induced and uninduced. Although AzoR-SC activity was the highest among all samples, it did not reach significant level. (B) The AzoR-SC activity against MR in crude cell lysate of cell culture, induced and uninduced. The enzyme activity of AzoR-SC reached 5000U/mL and it reached the significance of p<0.01.(C) The AzoR activity against RB5 in cell culture supernatant, induced and uninduced. The detected activity from induced samples were not significantly different form their corresponding control. (D) The AzoR-SC activity against RB5 in crude cell lysate of cell culture, induced and uninduced. The AzoR-SC activity reached the significance of p<0.05.
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.