Part:BBa_K2378006

NhaR-PETase Constitutive Coding Device

This is an optimized coding sequence of PETase enzyme for E. coli BL21 with the addition of NhaR BBa_K1357002 (https://parts.igem.org/wiki/index.php?title=Part:BBa_K1357002) coding sequence. NhaR enhances the transcription of pgaABCD operon which in turn induces the production of biofilm. This part also uses constitutive promoters BBa_J23106.

Crystal Violet Biofilm Assay

Expression of NhaR induces rapid production of biofilm. In order to observe the effect of NhaR in this part, we tested the rate of biofilm production of bacteria transformed with this part containing NhaR compared to control bacteria without NhaR.

The experiment was done by growing bacterial cultures in a 96-well microtiter plate, and measuring its biofilm formation after 6, 12, 18, 24 hours of incubation. The complete protocol can be seen here (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182663/).

The result shows a more rapid biofilm production in cultures transformed with this part containing NhaR compared to its control without NhaR. We can thus conclude that the function of this part to enhance biofilm production works as intended.

Figure 1. Quantification of biofilm production over 24 hours incubation of BBa_K2378006 transformants (containing NhaR) compared to its control

SEM (Scanning Electron Microscopy) Analysis

In order to observe the ability of our bacteria (transformed with BBa_K2378006) 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_K2378006 transformants, having access to PETase gene, compared to its control. We can thus conclude that BBa_K2378006 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 K2378006 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 1002
  Illegal NotI site found at 1911
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1656
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 236