Part:BBa_K1951008:Design

High level flagellin expression: This biobrick is composed of two parts

• BBa_K880005 design A strong promoter, strong RBS combination specifically conceived and tested for high expression levels of proteins in E.coli (this part is itself composed of sub-parts BBa_J23100 and BBa_B0034
• BBa_K1951005 Design the FliC coding sequence designed by our team.

Optimization of Bba_K1951008

Sequence optimization

We took fliC sequence from Glasgow 2014 team BBa_K1463601. We used Snapgene to remove the forbidden site by changing in the substitution which didn't change the amino acid. The sequence has been sythetised by IDT. We changed a Bbs1 site 130 position by substitution of amino acid coding.

Optimized codons 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 E. coli slic forward	cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGGCACAAGTCATTAAT
FliC E.coli slic reverse	ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAACCCTGGAGCAG

.

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1285
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 362
  Illegal AgeI site found at 770
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design of promotor BBa_J23100

Parts J23100 are a family of constitutive promoter parts isolated from a small combinatorial library. This promoter part can be used to tune the expression level of constitutively expressed parts. The NheI and AvrII restriction sites present within these promoter parts make them a scaffold for further modification.

Design of RBS BBa_B0034

Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix.

No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS.

Contact info for this part:

Primers: Note: EcoRI sites removed. Must use XP. Base pairs in CAPS are the truncated prefix and the suffix. Sequences provided by Gingko BioWorks.

Fwd :	GCTTCTAGAGATACATGAACATGCAATACttgacggctagctcagtcctaggtacagtgctagc
Rv :	CTGCAGCGGCCGCTACTAGTAGAGAGCGTTCtataaacgcagaaaggcccacccg

.

Source

We made this part from 2 others :

- BBa_K1951005

- BBa_K880005