Part:BBa_K5401008

RNAPAnc119

Ancestral sequence reconstruction has been a long-standing technique used in evolutionary biology to infer sequences of ancient proteins based on existing sequences. By using ancestral reconstruction, we are able to generate functional distant homologs through in silico methods. A programme called Molecular Evolutionary Genetics Analysis version X (MEGAX) was utilised for this purpose.

The ancestral sequence (RNAPAnc119) was inferred after a series of workflow. The ancestral sequence was subsequently cloned into stable T7-expressing plasmid (plasmid 1c) for downstream testing.

Usage and Biology

The ancestral sequence (RNAPAnc119) was derived based of using T7 RNA polymerase as the selection marker.

Characterization

The ancestral sequence (RNAPAnc119) was first transformed in our competent E. coli reporter cells harbouring plasmid C3 for evaluation of its efficiency. The bacterial culture was sub-cultured and subsequently induced with IPTG. 6 technical measurements were taken at 4 times point after induction - 10min, 30min, 60min and 120min, and once at 120min for negative (no induction).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1195
  Illegal EcoRI site found at 1927
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1195
  Illegal EcoRI site found at 1927
  Illegal NotI site found at 1141
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1195
  Illegal EcoRI site found at 1927
  Illegal BamHI site found at 1918
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1195
  Illegal EcoRI site found at 1927
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1195
  Illegal EcoRI site found at 1927
  
• 1000
  COMPATIBLE WITH RFC[1000]