Part:BBa_K5387001:Design

Design Notes

The sequence has been optimized for expression in E. coli.

The vector used was pET-28a(+), which His-tag we planned to utilized when expressing our protein.

The sequence encodes the amino acid sequence "QPPP", which is very difficult for the ribosomes in E. coli to translate [1]. We did not do it, but for future projects we would recommend altering this part of the sequence, either by mutation or when ordering the sequence.

Primers for cloning into pET-28a(+) between restriction sites NdeI and EcoRI:

fwd: 5'-GCGCCATATGGCTGTTTATAGGCTATGTGTAACTACAGGACCG-3'

rev: 5'-GCGCGAATTCTTAGATGCTAACGCTGTTCTCAATCAGTGGTGGG-3'

References

[1]: Qi F, Motz M, Jung K, Lassak J, Frishman D. Evolutionary analysis of polyproline motifs in Escherichia coli reveals their regulatory role in translation. PLoS Comput Biol. 2018 Feb 1;14(2):e1005987. doi: 10.1371/journal.pcbi.1005987. PMID: 29389943; PMCID: PMC5811046.