Part:BBa_K2378005

Constitutive PETase Coding Device

This is optimized coding sequence of PETase enzyme for E. coli BL21 with the addition of constitutive promoter BBa_J23106.

PETase Activity Assay (pNPB Assay)

PETase activity was tested using pNPB (p-nitrophenyl butyrate) Assay which measures esterase activity. Trasformants of BBa_K2378005 were grown for 4, 16, 24 hours in LB and tested against pNPB to determine their esterase activities, which were measured spectrophotometrically. As controls, we grew BL21 cells without plasmid with the same variations of age.

The results clearly showed that PETase activities were observed in all different variations of bacterial age, as the absorbance differences with their individual controls were significant. We can thus conclude that this part works as intended.

Figure 1. PETase Activity Assay (pNPB Assay) of BBa_K2378005 Transformants

The difference of PETase activity among the 3 varying culture ages were not significant. One major observation point is that in culture grown for only 4 hours, the initial activities were lower. This might be due to the that the initial concentration of available PETase was lower as well.

SEM (Scanning Electron Microscopy) Analysis

In order to observe the ability of our bacteria (transformed with BBa_K2378005) to degrade PET plastic via PETase gene embedded in this part, we ran a qualitative SEM analysis. PET plastic bottles were cut into 1x1 cm fragments with uniform weights. The plastic fragments were then washed with ethanol and water, followed by incubation in LB media contaning the bacterial transformants for 2 days. Following that, the plastic fragments were once again washed, dried, and finally observed under Scanning Electron Microscope (SEM). As the control, the plastic fragments were treated with bacterial cells without plasmid containing PETase.

SEM results show significantly rougher surface with more cracks in samples treated with BBa_K2378005 transformants, having access to PETase gene, compared to its control. We can thus conclude that BBa_K2378005 works as intended in biodegrading PET plastics.

SEM results show significantly rougher surface with more cracks in samples treated with BBa_K2378006 transformants, having access to PETase gene, compared to its control. We can thus conclude that BBa_K2378005 works as intended in biodegrading PET plastics.

Figure 2. SEM result of plastic fragment incubated with control BL21 cells bearing no plasmid

Figure 3. SEM result of plastic fragment incubated with BL21 cells transformed with BBa K2378005 shows rougher surface with more cracks

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NotI site found at 70
  Illegal NotI site found at 979
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 724
  
• 1000
  COMPATIBLE WITH RFC[1000]