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).

Alphafold was utilized to first predict the structure of all ancestral sequences of interest, providing insights into the protein's folding and functional domains. PyMOL was also employed to visualize the predicted structure, allowing for a detailed comparison with the wild-type and the RMSD value. After confirming the feasiblity of the structure through computational analyses, the ancestral sequences were then cloned into our stable T7-expressing plasmid, plasmid 1c, (see Engineering Success) for downstream testing and analysis.

Fig 1: Predicted structure for ancestral sequences of interest using Alphafold. [TOP] Alphafold predicted structures for all 3 ancestral sequences; and compared against the structure of wild-type T7RNAP (cyan). [BOTTOM LEFT] Sequence coverage of RNAPAnc119. [BOTTOM CENTER] Sequence coverage of RNAPAnc137. [BOTTOM RIGHT] Sequence coverage of RNAPAnc302.

The ancestral sequences were subsequently cloned into plasmid 1c using Gibson assembly, replacing the wild-type T7RNAP sequence. Purified plasmids were then transformed into competent E. coli reporter cells to compare their efficiency against the wild-type for an initial screening. Cells were then sub-cultured, followed by IPTG induction. Comparing the different variables tested, it is evident that the wild-type T7RNAP expressed from a plasmid (Stbl3 - 1c/C3) has shown the highest relative fluorescence intensity among all, higher than that observed by BL21 (DE3) cells transformed with the reporter plasmid. Comparing both the ancestral sequences (RNAPAnc119 and RNAPAnc137), little to no fluorescence intensity was similar, showing that the derived ancestral sequences have much lower processivity than the wild-type. As such, more developmental effots to improve the processitivity of the ancestral sequences are required before a comparable efficiency is observed.

Figure 2: Comparison of fluorescence measured at different time point after IPTG induction (10min, 30min, 60min, 120min). 6 technical measurements were taken for each sample.

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]