Difference between revisions of "Part:BBa K5387001:Design"
 
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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 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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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:  
+
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'  
 
: fwd: 5'-GCGCCATATGGCTGTTTATAGGCTATGTGTAACTACAGGACCG-3'  
 
: rev: 5'-GCGCGAATTCTTAGATGCTAACGCTGTTCTCAATCAGTGGTGGG-3'
 
: rev: 5'-GCGCGAATTCTTAGATGCTAACGCTGTTCTCAATCAGTGGTGGG-3'
  
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==References===
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==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.
 
[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.