Part:BBa_K1442101:Design
RdRP Mutant
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 492
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Principally, our goal with this part was to create a non-functioning version of our RdRP which we could test it against, to ensure it was the RdRP proper that was having the desired effect of replicating our RNA.
The same design notes are applicable to mutant RdRp (1-3) however the mutatation descriptor is found below:
To test the RdRP against a control, we used a deliberately mutated version of itself. To mutate it, we used the restriction enzyme BgLII (code: agatct). This was not thought through but arbitrarily chosen, and resulted in a shift of frame and the introduction of a stop codon (tga). Therefore, the region beyond said codon remains untranslated, so the phenotype of this mutant is determined entirely by the region previous to the stop codon.
1. The 21 amino acid residues have been removed in the part sequence, ensuring cytoplasmic RdRp activity, this ensures no anchoring to the endoplasmic reticulum in human cells (Lai et al., 2004). This modification has no major effect on RdRp activity, which is complimented by previous analysis showing no significant loss of nucleotide polymerization activity (Vo et al., 2004).
2. A single point mutation has been introduced at position 2884, where a guanine has replaced a cytosine. This confers an Arginine to glycine substitution, which increases the number of transformed colonies obtained (Bartenschlager et al., 2001).
3. A PstI restriction site was removed in the part sequence (CTGCAG) and was altered to CTCCAG to allow bio brick compatibility. This was necessary, as this would have invalidated this part as permissible for iGEM's RFC25 standard.
Source
The source from which this mutant is derived is our RdRP proper, part BBa_K1442100.