Difference between revisions of "Part:BBa K5401008"

Latest revision as of 10:58, 2 October 2024

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

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.

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. 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). 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.