Part:BBa_K3349005

Pectin Lyase PelC-GFP

The PelC Pectin Lyase is originally from Paenabacillus amylolyticus. This particular pectin lyase is more specific to methylated pectin or homogalacturonan substrates rather than polygalacturonic acid [1]. This protein also works with both PelB (BBa_K3349001) and Pnl (BBa_K3349001) in this organism to effectively break down pectin. PelC has an optimal activity at 55°C and a pH of 10. The Km in in terms of 80% methylated substrates is 0.41 +/-0.06mg/ml. Calcium is a cofactor for this protein and is therefore necessary within the buffer conditions [1].

A GFP reporter was added onto the C-terminus. As well a C-terminal his tag was added to the GFP for nickel affinity purification.

For more information on our design and use please see our webpage https://2020.igem.org/Team:Lethbridge_HS or visit the PelC gene part page BBa_K3349002.

Figure 1: Restriction digest of PelB and PelC DNA from the pUC57 plasmid using EcoRI and PstI enzymes run on a 1% agarose gel. The red boxes show the band corresponding to that of our DNA constructs. PelC-GFP in lane 4 shows similar results as well as an unexpected band at around 1500bp. We are not sure why this is occuring as there are no illegal cut sites within the sequence.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 829
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 215