Difference between revisions of "Part:BBa K398406:Design"
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===Design Notes=== | ===Design Notes=== | ||
− | + | The original sequences can be found in [http://www.ncbi.nlm.nih.gov/nuccore THIS LINK], these sequences were enhanced for their expression on ''E. coli'' K12. Additionally, the restriction sites for the enzymes XbaI, PstI, EcoRI and SpeI were removed from the CDS and the stop codon was replaced by the codon TAA in order to avoid the formation of possible ORF's. For this process we used the [http://www.jcat.de/ jcat] software tool. | |
+ | An extra XbaI site can be found between RBS and Promoter so you can increase the expression level of the protein by changing the promoter. | ||
+ | |||
+ | The expected size for Prefoldin alpha-subunit is 16.97 kDa, its aminoacid sequence can be checked below: | ||
+ | |||
+ | 1 mirmaqnnke leklayeyqv lqaqaqilaq nlellnlaka evqtvretle nlkkieeekp | ||
+ | 61 eilvpigags flkgvivdkn naivsvgsgy aversideai sflekrlkey deaikktqga | ||
+ | 121 laelekrige varkaqevqq kqsmtsfkvk k | ||
+ | |||
+ | The expected size for Prefoldin beta-subunit is 13.28 kDa, its aminoacid sequence can be checked below: | ||
+ | |||
+ | 1 mqnippqvqa mlgqldtyqq qlqlviqqkq kvqadlneak kaleeietlp ddaqiyktvg | ||
+ | 61 tlivkttkek avqelkekie tlevrlnaln rqeqkinekv keltqkiqaa lrpptag | ||
===Source=== | ===Source=== | ||
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Genomic sequence obtained from NCBI. | Genomic sequence obtained from NCBI. | ||
− | Prefoldin alpha accession number: | + | Prefoldin alpha accession number: [http://www.ncbi.nlm.nih.gov/gene/1444414 PH0527] |
+ | |||
Prefoldin beta accession number: [http://www.ncbi.nlm.nih.gov/gene/1444421 PH0532] | Prefoldin beta accession number: [http://www.ncbi.nlm.nih.gov/gene/1444421 PH0532] | ||
===References=== | ===References=== | ||
+ | #Okochi M. ''et al.'', "Overexpression of prefoldin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 endowed Escherichia coli with organic solvent tolerance", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, Volume 79, Number 3, 443-449, DOI: 10.1007/s00253-008-1450-1. | ||
+ | #Tanaka T. ''et al.'', Direct submission of the sequence. (11-JUN-1998) Biotechnology Center, National Institute of Technology and Evaluation, 2-49-10 Nishihara, Shibuyaku, Tokyo 151-0066, Japan. |
Latest revision as of 16:59, 27 October 2010
Solvent tolerance cluster
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 37
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 37
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 37
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The original sequences can be found in [http://www.ncbi.nlm.nih.gov/nuccore THIS LINK], these sequences were enhanced for their expression on E. coli K12. Additionally, the restriction sites for the enzymes XbaI, PstI, EcoRI and SpeI were removed from the CDS and the stop codon was replaced by the codon TAA in order to avoid the formation of possible ORF's. For this process we used the [http://www.jcat.de/ jcat] software tool.
An extra XbaI site can be found between RBS and Promoter so you can increase the expression level of the protein by changing the promoter.
The expected size for Prefoldin alpha-subunit is 16.97 kDa, its aminoacid sequence can be checked below:
1 mirmaqnnke leklayeyqv lqaqaqilaq nlellnlaka evqtvretle nlkkieeekp 61 eilvpigags flkgvivdkn naivsvgsgy aversideai sflekrlkey deaikktqga 121 laelekrige varkaqevqq kqsmtsfkvk k
The expected size for Prefoldin beta-subunit is 13.28 kDa, its aminoacid sequence can be checked below:
1 mqnippqvqa mlgqldtyqq qlqlviqqkq kvqadlneak kaleeietlp ddaqiyktvg 61 tlivkttkek avqelkekie tlevrlnaln rqeqkinekv keltqkiqaa lrpptag
Source
Genomic sequence obtained from NCBI.
Prefoldin alpha accession number: [http://www.ncbi.nlm.nih.gov/gene/1444414 PH0527]
Prefoldin beta accession number: [http://www.ncbi.nlm.nih.gov/gene/1444421 PH0532]
References
- Okochi M. et al., "Overexpression of prefoldin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 endowed Escherichia coli with organic solvent tolerance", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, Volume 79, Number 3, 443-449, DOI: 10.1007/s00253-008-1450-1.
- Tanaka T. et al., Direct submission of the sequence. (11-JUN-1998) Biotechnology Center, National Institute of Technology and Evaluation, 2-49-10 Nishihara, Shibuyaku, Tokyo 151-0066, Japan.