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.

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

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