Team:ITB Indonesia/Improve


Improve


Improve

BBa_K2378007 is an improved coding sequence of IrrE BBa_K729001 from by Team UCL London 2012. When transformed to E. coli, this protein protected from salt, oxidative, and thermal shock. Background of Improvement We tried to analyse existing IrrE part from BBa_K729001 Team UCL London, and found some possible error in the coding sequence. The translated nucleotide is not a contiguous protein, which is shown by a stop codon in the middle of the coding sequence, continued by another start codon ~90 base downstream. At first, we thought that it's the IrrE nature to have two separate active proteins. But after we find the corresponding Uniprot accession number, there are important domain of protein in the 75-199 position of the residues, under the classification of peptidases. This domain is essential for the activity of IrrE functionality because it cleaves and inactivates repressor protein DdrO, leading to the upregulation of several DNA repair and other genes after exposure of the cells to radiation.[1] From the alignment process, it is shown that the 'lost' protein from the stop codon is not from the peptidase domain sequence. But, from the length of the untranslated residues (~30 aa), it is hypothesized that this will affect the structural stability of the protein itself. Moreover, the second protein is not functional and does not have any motifs, hence will be possible burden for the metabolic process efficiency.

Improvement

We optimized the sequence for the expression in Escherichia coli BL21 by using synonymous mutations, then made the protein contiguous while being translated. We also removed the low complexity region.

Figure 1: Protein architecture of BBa_K729001. DUF955 is peptidase domain.
Figure 2: Protein architecture of novel BBa K2378007. DUF955 is peptidase domain.

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