Part:BBa_K2378004
pSal + PETase Coding Device
ITB_Indonesia iGEM team 2017 designed a PETase enzyme coding device consisting of NahR-pSal regulatory part (BBa_J61051, https://parts.igem.org/Part:BBa_J61051) which is commonly activated by salicylate induction. Besides salicylate, NahR-pSal regulatory system is also known to have a certain degree of responsiveness in the presence of organic pollutants (OPs). This particular characteristic of NahR-pSal is utilized in iGEM 2017 Indonesia's project to detect the presence of plastic.
PETase Activity (pNPB Assay) and pSal Inducibility Assay
PETase activity was tested using pNPB (p-nitrophenyl butyrate) Assay which measures esterase activity. Trasformants of K2378004 were grown in LB and tested against pNPB to determine their esterase activities, which were measured spectrophotometrically. As controls, we grew BL21 cells without plasmid.
To test NahR-pSal regulatory system's inducibility, we induced the expression of PETase (measured using pNPB Assay) in two different ways; 1mM of salicylate, compared to direct use of organic pollutants from plastic surface as inducers.
The results showed that PETase expression can be induced using 1mM of salicylate. However, the the inducibility of NahR-pSal system using organic pollutants from plastic surface could not be determined due to the fact that although the expression PETase can be observed, the trend is not significantly different from uninduced culture. This is due to the fact that NahR-pSal regulatory system used might be leaky to a certain degree.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 1303
Illegal NotI site found at 2212 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 786
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 77
Illegal NgoMIV site found at 618
Illegal AgeI site found at 1957 - 1000COMPATIBLE WITH RFC[1000]
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