Difference between revisions of "Part:BBa K2378004"
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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.
 
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.
  
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<p style="font-size: 18px"><strong>PETase Activity (pNPB Assay) and pSal Inducibility Assay </strong></p>
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<p>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.</p>
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<p>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.</p>
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<p>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.</p>
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<p>[[File:T--ITB Indonesia--psalpnpb.jpeg|400px|thumb|center|Figure 1. PETase Activity Assay (pNPB Assay) of BBa K2378004 Transformants]]</p>
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Revision as of 21:38, 1 November 2017


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.

Figure 1. PETase Activity Assay (pNPB Assay) of BBa K2378004 Transformants

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 1303
    Illegal NotI site found at 2212
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 786
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 77
    Illegal NgoMIV site found at 618
    Illegal AgeI site found at 1957
  • 1000
    COMPATIBLE WITH RFC[1000]