Part:BBa_K4807022

Catalase insert

In order to achieve the overexpression of rhtA exporter and Human catalase, we employed pETDuet-1 (BBa_J100519)as our carry out vector. This vector features a ColE1/pMB1/pBR322/pUC origin of replication and it is ampicillin resistant.

pETDuet-1 is a high copy number plasmid that features two T7 promoters and two RBS sequences. However, it contains only one T7 terminator. As a result, each gene is expressed under its dedicated T7 promoter, but one of the inserted genes requires the inclusion of a T7 terminator (BBa_K4807002) for proper regulation. The rhtA insert (BBa_K4807023) is the one carrying the extra T7 terminator.

In order to assemble our constructs, two cloning steps needed to be performed. Firstly, the vector was linearized through digestion with the BglII restriction enzyme to allow the insertion of the CAT gene (BBa_K4807003). Subsequently, another digestion with SacI was necessary to clone the rhtA exporter gene (BBa_K1977000) into our vector.

In order to confirm our gene expression, an S-tag (BBa_K3630004) was incorporated in the catalase insert making it possible to detect through Western-Blotting.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 248
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 248
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 248
  Illegal BamHI site found at 60
  Illegal BamHI site found at 513
  Illegal BamHI site found at 576
  Illegal XhoI site found at 1187
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 248
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 248
  Illegal NgoMIV site found at 1435
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1528