Difference between revisions of "Part:BBa K1951008:Design"
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We made this part from 2 others : | We made this part from 2 others : | ||
| − | - [https://parts.igem.org/Part:BBa_K1951005 | + | - [https://parts.igem.org/Part:BBa_K1951005 BBa_K1951005] |
- [https://parts.igem.org/Part:BBa_K880005 BBa_K880005] | - [https://parts.igem.org/Part:BBa_K880005 BBa_K880005] | ||
===References=== | ===References=== | ||
Revision as of 14:31, 15 October 2016
High flagellin expression coding sequence has been designed using two parts :
- BBa_K880005 design Strong promoter, strong RBS combination for high expression levels of proteins (composed of sub-parts design BBa_J23100 and design BBa_B0034
- BBa_K1951005 Design the FliC coding sequence designed by our team
Contents
Optimization
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[1]. 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[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 E. coli slic forward | cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGGCACAAGTCATTAAT |
| FliC E.coli slic reverse | ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAACCCTGGAGCAG |
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1285
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 362
Illegal AgeI site found at 770 - 1000COMPATIBLE WITH RFC[1000]
Promotor design
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.
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 :
References
- ↑ http://eu.idtdna.com/site
- ↑ https://eu.idtdna.com/CodonOpt