Difference between revisions of "Part:BBa K5387001:Design"
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===Design Notes=== | ===Design Notes=== | ||
The sequence has been optimized for expression in ''E. coli''. | The sequence has been optimized for expression in ''E. coli''. | ||
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| + | 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. | 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. | ||
Latest revision as of 10:32, 29 September 2024
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.