Difference between revisions of "Part:BBa K1951006:Design"

 
 
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===Design Notes===
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We used Snapgene to remove the forbidden site,  we modified Pst1 site CTGCAG into CTGCGG,  by changing in the substitution which didn't change the amino acid. The sequence has been sythetised by IDT<ref>http://eu.idtdna.com/site</ref>.
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We also added prefix and suffix sequences containing restriction sites EcoRI, XbaI and SpeI, PstI respectively.
  
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__NOTOC__
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===Codon optimization for <i>Escherichia coli</i>. ===
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<partinfo>BBa_K1951006 short</partinfo>
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We obtimised codon for <i>Escherichia coli</i>. Codon optimization is a technique used to <u>improve the protein expression</u> in living organism by increasing the translational efficiency of gene of interest [1-4, 6-13, 15-18, 20-28]. This biobrick codon optimised <u>increase the functionality of gene</u>. To process it, we use codon optimization IDT software<ref> https://eu.idtdna.com/CodonOpt </ref>.
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If you use this biobrick in <i>Escherichia coli</i>, you can be sure that the protein produced will be highly expressed and well solubilised.
  
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<partinfo>BBa_K1951006 SequenceAndFeatures</partinfo>
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=== Prefix and suffix addition===
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===Design Notes===
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We modified of the site Pst1 CTGCAG into CTGCGG.
+
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We added prefix and suffix subsequences containing EcoRI, XbaI and SpeI, PstI respectively.
+
  
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Prefix and suffix subsequences (containing restriction site EcoRI, XbaI and SpeI PstI respectively) have been added by a SLIC method with the following oligos :
  
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{| class="wikitable"
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| FliC ''D. vulgaris'' slic forward 
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| cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGTCACTGGTTATCAAT
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|-
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| FliC ''D. vulgaris'' slic reverse
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| ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAGCCGCCGAGAAG
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|}.
  
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== Sequences and features==
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<partinfo>BBa_K1951006 SequenceAndFeatures</partinfo>
  
 
===Source===
 
===Source===
  
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DNA sequence has been taken from the following website :  
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The coding sequence has been found on the following website :  
 
http://www.ncbi.nlm.nih.gov/nucleotide/46451220?report=genbank&log$=nucltop&blast_rank=3&RID=WDB55ADB014  
 
http://www.ncbi.nlm.nih.gov/nucleotide/46451220?report=genbank&log$=nucltop&blast_rank=3&RID=WDB55ADB014  
  
 
===References===
 
===References===

Latest revision as of 12:51, 17 October 2016

Design Notes

We used Snapgene to remove the forbidden site, we modified Pst1 site CTGCAG into CTGCGG, by changing in the substitution which didn't change the amino acid. The sequence has been sythetised by IDT[1]. We also added prefix and suffix sequences containing restriction sites EcoRI, XbaI and SpeI, PstI respectively.

Codon optimization for Escherichia coli.

We obtimised codon for Escherichia coli. Codon optimization is a technique used to improve the protein expression in living organism by increasing the translational efficiency of gene of interest [1-4, 6-13, 15-18, 20-28]. This biobrick codon optimised increase the functionality of gene. To process it, we use codon optimization IDT software[2]. If you use this biobrick in Escherichia coli, you can be sure that the protein produced will be highly expressed and well solubilised.

Prefix and suffix addition

Prefix and suffix subsequences (containing restriction site EcoRI, XbaI and SpeI PstI respectively) have been added by a SLIC method with the following oligos :

FliC D. vulgaris slic forward cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGTCACTGGTTATCAAT
FliC D. vulgaris slic reverse ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAGCCGCCGAGAAG
.

Sequences and features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 301
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 400

Source

The coding sequence has been found on the following website : http://www.ncbi.nlm.nih.gov/nucleotide/46451220?report=genbank&log$=nucltop&blast_rank=3&RID=WDB55ADB014

References

  1. ↑ http://eu.idtdna.com/site
  2. ↑ https://eu.idtdna.com/CodonOpt