Difference between revisions of "Part:BBa K1951009:Design"
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===Design Notes=== | ===Design Notes=== | ||
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===Sequence optimization=== | ===Sequence optimization=== | ||
| − | We found the raw sequence from online data base | + | We found the raw sequence from online data base [http://www.ncbi.nlm.nih.gov/nucleotide/46451220?report=genbank&log$=nucltop&blast_rank=3&RID=WDB55ADB014 see here]. We removed all illegal sites (EcoRI, XbaI, PstI SpeI) and BbsI. Then we optimized the codon use for expression in''E. coli'',with an online [https://eu.idtdna.com/CodonOpt codon optimizer]. As the optimization is random, some illegal or BbsI sites has appeared and we did the optimization until obtain a sequence with no such sites. |
The sequence amplified from the synthetized one by IDT was amplified and cloned into the pSB1C3 vector thanks to SLIC primers designed by our team. | The sequence amplified from the synthetized one by IDT was amplified and cloned into the pSB1C3 vector thanks to SLIC primers designed by our team. | ||
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===Promoter and RBS selection=== | ===Promoter and RBS selection=== | ||
| − | As the purpose of this part is high level protein expression we selected the part [https://parts.igem.org/Part: | + | As the purpose of this part is high level protein expression we selected the part [https://parts.igem.org/Part:BBa_K880005 BBa_K880005] to drive expression of our coding sequence. We chose this part as: |
| + | * it has already been successfully used in the past, | ||
| + | * it was available in the distribution, | ||
* it combines a strong promotor and a RBS (so avoiding the separate incorporation of 2 parts). | * it combines a strong promotor and a RBS (so avoiding the separate incorporation of 2 parts). | ||
===Source=== | ===Source=== | ||
| − | We | + | We constructed this part from 2 others : |
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| − | + | * [https://parts.igem.org/Part:BBa_K1951006 BBa_K1951006] | |
| + | * [https://parts.igem.org/Part:BBa_K880005 BBa_K880005] | ||
Latest revision as of 14:31, 17 October 2016
Design Notes
High level flagellin expression: This biobrick is composed of two parts:
- BBa_K880006 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 the FliC coding sequence designed by our team for high level expression in Desulfovibrio vulgaris with sequence optimization.
Sequence optimization
We found the raw sequence from online data base [http://www.ncbi.nlm.nih.gov/nucleotide/46451220?report=genbank&log$=nucltop&blast_rank=3&RID=WDB55ADB014 see here]. We removed all illegal sites (EcoRI, XbaI, PstI SpeI) and BbsI. Then we optimized the codon use for expression inE. coli,with an online codon optimizer. As the optimization is random, some illegal or BbsI sites has appeared and we did the optimization until obtain a sequence with no such sites.
The sequence amplified from the synthetized one by IDT was amplified and cloned into the pSB1C3 vector thanks to SLIC primers designed by our team.
Promoter and RBS selection
As the purpose of this part is high level protein expression we selected the part BBa_K880005 to drive expression of our coding sequence. We chose this part as:
- it has already been successfully used in the past,
- it was available in the distribution,
- it combines a strong promotor and a RBS (so avoiding the separate incorporation of 2 parts).
Source
We constructed this part from 2 others :