Difference between revisions of "Part:BBa K5387001:Design"
 
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===Design Notes===
 
===Design Notes===
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The sequence has been optimized for expression in E. coli.  
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The sequence has been optimized for expression in ''E. coli''.  
  
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The sequence contains the amino acid sequence "QPPP", which is very difficult for the ribosomes in E. coli to translate KÄLLA. 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.
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The vector used was pET-28a(+), which His-tag we planned to utilized when expressing our protein.  
  
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Primers for cloning into pET28a(+) between restriction sites NdeI and EcoRI:
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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.
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fwd: 5'-GCGCCATATGGCTGTTTATAGGCTATGTGTAACTACAGGACCG-3'
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rev: 5'-GCGCGAATTCTTAGATGCTAACGCTGTTCTCAATCAGTGGTGGG-3'
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===Source===
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Primers for cloning into pET-28a(+) between restriction sites NdeI and EcoRI:
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This gene is expressed in humans KÄLLA
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: fwd: 5'-GCGCCATATGGCTGTTTATAGGCTATGTGTAACTACAGGACCG-3'
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: rev: 5'-GCGCGAATTCTTAGATGCTAACGCTGTTCTCAATCAGTGGTGGG-3'
  
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==References==
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==References===
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[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.

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.