Part:BBa_K1951006:Design

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. 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. 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

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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