Difference between revisions of "Part:BBa K1951008:Design"
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| + | ===Design Notes=== | ||
| − | + | High level flagellin expression: | |
| − | < | + | This biobrick is composed of two parts: |
| + | * [https://parts.igem.org/Part:BBa_K880005 BBa_K880005] A strong promoter, strong RBS combination specifically conceived and tested for high expression levels of proteins in <i>E.coli</i> (this part is itself composed of sub-parts [https://parts.igem.org/Part:BBa_J23100 BBa_J23100 ] and [https://parts.igem.org/Part:BBa_B0034 BBa_B0034] | ||
| + | * [https://parts.igem.org/Part:BBa_K1951005 BBa_K1951005] the FliC coding sequence designed by our team for high level expression in <i>E.coli</i> with sequence optimization. | ||
| − | |||
| + | == Optimization of Bba_K1951008== | ||
| − | === | + | ===Sequence optimization=== |
| − | + | ||
| + | This part is optimized for expression in <i>E.coli</i> by codon optimization and synthesis of a synthetic gene. | ||
| + | Codon optimization was done using the tool provided by IDT <ref> https://eu.idtdna.com/CodonOpt </ref>. | ||
| + | Codon optimization has been shown to <u>improve protein expression</u> by increasing the translational efficiency of gene of interest, and consists of choosing for each amino acid the codon that works best in the target species <b>Claire a ref or 2</b>. | ||
| + | We optimized the sequence for expression in <i>E.coli</i>. | ||
| + | In addition to changing the codons from the <i>E.coli</i> sequence we also ensured that all forbidden restriction sites were absent to ensure compatibility with the biobrick standard. | ||
| + | We also designed the insertion of a Bbs1 site at amino acid 130 to allow insertion of additional sequences in the loop at this position in the sequence. Unfortunately we did not get round to using this feature. | ||
| − | === | + | ===Promoter and RBS selection=== |
| − | We | + | 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). | ||
| + | |||
| + | |||
| + | <partinfo>BBa_K1951008 SequenceAndFeatures</partinfo> | ||
| + | |||
| + | ===Source=== | ||
| − | + | We constructed this part from 2 others : | |
| − | + | * [https://parts.igem.org/Part:BBa_K1951005 BBa_K1951005] | |
| + | * [https://parts.igem.org/Part:BBa_K880005 BBa_K880005] | ||
===References=== | ===References=== | ||
Latest revision as of 14:30, 17 October 2016
Contents
Design Notes
High level flagellin expression: This biobrick is composed of two parts:
- BBa_K880005 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 E.coli with sequence optimization.
Optimization of Bba_K1951008
Sequence optimization
This part is optimized for expression in E.coli by codon optimization and synthesis of a synthetic gene. Codon optimization was done using the tool provided by IDT [1]. Codon optimization has been shown to improve protein expression by increasing the translational efficiency of gene of interest, and consists of choosing for each amino acid the codon that works best in the target species Claire a ref or 2. We optimized the sequence for expression in E.coli.
In addition to changing the codons from the E.coli sequence we also ensured that all forbidden restriction sites were absent to ensure compatibility with the biobrick standard. We also designed the insertion of a Bbs1 site at amino acid 130 to allow insertion of additional sequences in the loop at this position in the sequence. Unfortunately we did not get round to using this feature.
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).
- 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]
Source
We constructed this part from 2 others :